Compositions and methods for improving tomato production

ABSTRACT

The present invention provides both compositions comprising  Methylobacterium  and compositions comprising  Methylobacterium  that are depleted of substances that promote growth of resident microorganisms on a tomato plant or seed. Also provided are methods for improving tomato production, methods of making the compositions, and methods of treating a tomato plant, plant part, or seed with the compositions comprising  Methylobacterium.

CROSS REFERENCE TO RELATED APPLICATIONS

This International patent application claims the benefit of U.S. Provisional Patent Application No. 61/954,390, filed Mar. 17, 2014, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING STATEMENT

A sequence listing containing the file named 53907-138691_SL.txt which is 14,824,679 bytes (measured in MS-Windows®) and created on Dec. 3, 2014, comprises 9,188 sequences, is provided herewith via the USPTO's EFS system, and is incorporated herein by reference in its entirety.

BACKGROUND

One-carbon organic compounds such as methane and methanol are found extensively in nature, and are utilized as carbon sources by bacteria classified as methanotrophs and methylotrophs. Methanotrophic bacteria include species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and Methylocella (Lidstrom, 2006). Methanotrophs possess the enzyme methane monooxygenase, that incorporates an atom of oxygen from O₂ into methane, forming methanol. All methanotrophs are obligate one-carbon utilizers that are unable to use compounds containing carbon-carbon bonds. Methylotrophs, on the other hand, can also utilize more complex organic compounds, such as organic acids, higher alcohols, sugars, and the like. Thus, methylotrophic bacteria are facultative methylotrophs. Methylotrophic bacteria include species in the genera Methylobacterium, Hyphomicrobium, Methylophilus, Methylobacillus, Methylophaga, Aminobacter, Methylorhabdus, Methylopila, Methylosulfonomonas, Marinosulfonomonas, Paracoccus, Xanthobacter, Ancylobacter (also known as Microcyclus), Thiobacillus, Rhodopseudomonas, Rhodobacter, Acetobacter, Bacillus, Mycobacterium, Arthobacter, and Nocardia (Lidstrom, 2006).

Most methylotrophic bacteria of the genus Methylobacterium are pink-pigmented. They are conventionally referred to as PPFM bacteria, being pink-pigmented facultative methylotrophs. Green (2005, 2006) identified twelve validated species in the genus Methylobacterium, specifically M. aminovorans, M. chloromethanicum, M. dichloromethanicum, M. extorquens, M. fujisawaense, M. mesophilicum, M organophilum, M. radiotolerans, M. rhodesianum, M. rhodinum, M. thiocyanatum, and M. zatmanii. However, M. nidulans is a nitrogen-fixing Methylobacterium that is not a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in nature, being found in soil, dust, fresh water, sediments, and leaf surfaces, as well as in industrial and clinical environments (Green, 2006).

SUMMARY

Provided herein are compositions comprising Methylobacterium that are depleted of substances that promote growth of resident bacteria on the plant or seed, compositions comprising a solid substance with adherent Methylobacterium grown thereon or an emulsion having Methylobacterium grown therein, compositions comprising certain Methylobacterium isolates and derivatives thereof, methods of using the compositions to improve tomato production, and methods of making the compositions. Such compositions are in certain instances referred to herein as simply “Methylobacterium-containing compositions”. In certain embodiments, the Methylobacterium in the composition or that is used is strain NLS0037, a variant thereof, or a strain having polymorphic DNA markers present in NLS0037 that are absent from a strain that does not increase tomato seedling growth in comparison to an untreated control. In certain embodiments, the Methylobacterium in the composition or that is used is strain NLS0037 and the composition is used to treat a tomato seed. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 with the proviso that the gene is not found in M. extorquens AM1, M. extorquens PA1, or M. extorquens ME4. In certain embodiments, the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4. In certain embodiments, the Methylobacterium in the composition or that is used is a Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof. In certain embodiments, any of the aforementioned compositions can further comprise an agriculturally acceptable excipient, an agriculturally acceptable adjuvant, or combination thereof.

Methods for improving tomato production comprising applying a coating or partial coating of a composition comprising Methylobacterium to a tomato plant, a part thereof, or to a tomato seed, wherein said composition comprises a solid substance with adherent Methylobacterium grown thereon, an emulsion having Methylobacterium grown therein, or compositions comprising certain Methylobacterium isolates and derivatives thereof, and wherein said tomato plant or tomato plant grown from said seed exhibits a trait improvement selected from the group consisting of an increased rate of root growth, leaf growth, seedling growth, seed production, fruit production, scion production, rootstock production, and/or increased total biomass decreased cycle time, and combinations thereof when compared to an untreated control tomato plant or a control tomato plant grown from an untreated seed are provided herein. Methods comprising applying a composition comprising Methylobacterium to a tomato plant, a part thereof, or to a tomato seed, wherein said composition comprises: (i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion having Methylobacterium grown therein; (iii) a Methylobacterium that has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594; or (iv) a Methylobacterium selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof, and wherein said tomato plant or tomato plant grown from said seed exhibits a trait improvement selected from the group consisting of an increased rate of root growth, leaf growth, seedling growth, seed production, fruit production, scion production, rootstock production, and/or increased total biomass when compared to an untreated control tomato plant or a control tomato plant grown from an untreated seed, thereby obtaining improved tomato production, are also provided. In certain embodiments, the composition comprises Methylobacterium at a titer of about 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solid composition or at a titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL for a liquid composition containing the solid substance or for the emulsion. In certain embodiments, the Methylobacterium has at least one polymorphic DNA element that is present in Methylobacterium strain NLS0037 but that is absent from a strain that does not increase tomato seedling growth. In certain embodiments, the applied composition coats or partially coats said plant or a part thereof, or said seed. In certain embodiments, the composition is applied in a hydroponic solution. In certain embodiments, the methods further comprise: (i) growing said tomato plant or tomato plant grown from said seed; and/or (ii) harvesting seedlings, rootstock, scions, fruit, or seed from said tomato plant or tomato plant grown from said seed. In certain embodiments, the solid substance with adherent Methylobacterium is not a substance that promotes growth of resident microorganisms on the tomato plant, the part thereof, or the tomato seed. In certain embodiments, the composition comprises an agriculturally acceptable adjuvant and/or excipient. In certain embodiments of any of the aforementioned methods, the composition is depleted of substances that promote growth of resident microorganisms on said plant or seed. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 with the proviso that the gene is not found in M. extorquens AM1, M. extorquens PA1, or M. extorquens ME4. In certain embodiments, the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof. Also provided are tomato plant parts or tomato seeds obtained by any of the aforementioned methods and that are coated or partially coated with a composition comprising Methylobacterium.

Methods for improving tomato plant production comprising applying a composition comprising Methylobacterium to a tomato plant, a part thereof, or tomato seed, wherein said composition is depleted of substances that promote growth of resident microorganisms on said plant or seed and wherein said plant or plant grown from said seed exhibits a trait improvement selected from the group consisting of an increased rate of leaf growth, an increased rate of root growth, increased total biomass production, increased seed yield, decreased cycle time, and combinations thereof when compared to an untreated control tomato plant or a control tomato plant grown from an untreated seed. In certain embodiments, the composition comprises a solid substance with adherent Methylobacterium grown thereon. In certain embodiments, the solid substance is not a substance that promotes growth of resident microorganisms on the tomato plant, the part thereof, or the tomato seed. In certain embodiments, the composition comprises Methylobacterium at a titer of about 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm. In certain embodiments, the composition comprises a liquid, a solid substance with Methylobacterium adhered thereto in a liquid, a solid substance with Methylobacterium adhered thereto in an emulsion, or an emulsion. In certain embodiments, the composition comprises Methylobacterium at a titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL. In certain embodiments, the methods further comprise: (i) growing said tomato plant or tomato plant grown from said seed; and/or (ii) harvesting seedlings, rootstock, scions, fruit, or seed from said tomato plant or tomato plant grown from said seed. In certain embodiments, the Methylobacterium has at least one polymorphic DNA element that is present in at least one Methylobacterium strain selected from the group consisting of NLS0037 but that is absent from a strain that does not increase tomato seedling growth. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 with the proviso that the gene is not found in M. extorquens AM1, M. extorquens PA1, or M. extorquens ME4. In certain embodiments, the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof. In certain embodiments of any of the aforementioned methods, the composition coats or partially coats said plant or a part thereof, or said seed. In certain embodiments the tomato plant part or tomato seed is immersed or partially immersed in the composition. In certain embodiments of any of the aforementioned methods, the composition is applied in a hydroponic solution. Also provided are tomato plants, plant parts or tomato seeds obtained by any of the aforementioned methods and that are coated or partially coated with a composition comprising Methylobacterium.

Compositions comprising: (a) (i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion comprising Methylobacterium; or (iii) certain Methylobacterium sp. are provided. In certain embodiments, compositions comprising: (i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion with Methylobacterium grown therein or contained therein; or (iii) a Methylobacterium; wherein said Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 or wherein the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4, and wherein said composition further comprises an agriculturally acceptable adjuvant and/or excipient or wherein the composition comprises a hydroponic solution of man-made origin are provided. In certain embodiments, the compositions can comprise: (i) a solid substance with adherent Methylobacterium grown thereon or (ii) an emulsion with Methylobacterium grown therein or contained therein, wherein said Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 or wherein the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4, and wherein said composition further comprises an agriculturally acceptable adjuvant and/or excipient or wherein the composition comprises a hydroponic solution of man-made origin. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 with the proviso that the gene is not found in M. extorquens AM1, M. extorquens PA1, or M. extorquens ME4. In certain embodiments, the Methylobacterium has at least one polymorphic DNA element that is present in Methylobacterium isolate NLS0037. In certain embodiments, the Methylobacterium is NLS0037 a variant thereof, or a strain having polymorphic DNA markers present in NLS0037 that are absent from a strain that does not increase tomato seedling growth in comparison to an untreated control. In certain embodiments, the Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594. In certain embodiments the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof. In certain embodiments, the composition is depleted of substances that promote growth of resident microorganisms on a plant or seed. In certain embodiments, the substance that promotes growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorous source, a sulfur source, a magnesium source, and combinations thereof. In certain embodiments, the compositions further comprise an agriculturally acceptable adjuvant and/or excipient. In certain embodiments, the solid substance with adherent Methylobacterium grown thereon has a Methylobacterium titer of at least about 5×10⁸ CFU/gm to at least about 1×10¹⁴ CFU/gm. In certain embodiments, the aforementioned compositions are adapted for use in treating a tomato plant or seed or is used to treat a tomato plant or seed. Also provided herein is a tomato plant part or tomato seed that is coated or partially coated with any of the aforementioned compositions. Also provided herein is a tomato plant part or tomato seed that is immersed or partially immersed in any of the aforementioned compositions.

Also provided herein are methods of identifying compositions, plant parts, plant seeds, or processed plant products comprising Methylobacterium sp. NLS017 or NLS066 by assaying for the presence of nucleic acid sequences contained in SEQ ID NO: 4595-9188 in those materials. In certain embodiments, such methods can comprise subjecting a sample suspected of containing Methylobacterium sp. NLS017 or NLS066 to a nucleic acid analysis technique and determining that the sample contains one or more nucleic acid containing a sequence of at least about 20, 50, 100, 200, 500, or a 1000 nucleotides that is identical to at least one of SEQ ID NO: 4595-9188, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 4595-7278 is indicative of the presence of NLS017 and wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 7279-9188 is indicative of the presence of NLS066. Such nucleic acid analyses include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore based detection, branched DNA analyses, combinations thereof, and the like.

Also provided herein are methods of identifying Methylobacterium sp. that can confer useful traits to plants by assaying for the presence of nucleic acid sequences contained in SEQ ID NO: 4595-9188 in the Methylobacterium sp. In certain embodiments, such methods can comprise subjecting a candidate Methylobacterium sp. to a nucleic acid analysis technique and determining that the sample contains one or more nucleic acid containing a sequence of at least about 20, 50, 100, 200, 500, or a 1000 nucleotides that is identical to at least one of SEQ ID NO: 4595-9188 indicates that the candidate Methylobacterium sp. that can confer a useful traits to a plant. Such nucleic acid analyses include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore based detection, branched DNA analyses, combinations thereof, and the like.

DESCRIPTION Definitions

As used herein, the phrases “adhered thereto” and “adherent” refer to Methylobacterium that are associated with a solid substance by growing, or having been grown, on a solid substance.

As used herein, the phrase “agriculturally acceptable adjuvant” refers to a substance that enhances the performance of an active agent in a composition for treatment of plants and/or plant parts. In certain compositions, an active agent can comprise a mono-culture or co-culture of Methylobacterium.

As used herein, the phrase “agriculturally acceptable excipient” refers to an essentially inert substance that can be used as a diluent and/or carrier for an active agent in a composition for treatment of plants and/or plant parts. In certain compositions, an active agent can comprise a mono-culture or co-culture of Methylobacterium.

As used herein, the term “Methylobacterium” refers to bacteria that are facultative methylotrophs of the genus Methylobacterium. The term Methylobacterium, as used herein, thus does not encompass includes species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and Methylocella, which are obligate methanotrophs.

As used herein, the phrase “co-culture of Methylobacterium” refers to a Methylobacterium culture comprising at least two strains of Methylobacterium or at least two species of Methylobacterium.

As used herein, the phrase “contaminating microorganism” refers to microorganisms in a culture, fermentation broth, fermentation broth product, or composition that were not identified prior to introduction into the culture, fermentation broth, fermentation broth product, or composition.

As used herein, the phrase “derivatives thereof”, when used in the context of a Methylobacterium strain, refers to any strain that is obtained from the Methylobacterium strain. Derivatives of a Methylobacterium strain include, but are not limited to, variants of the strain obtained by selection, variants of the strain selected by mutagenesis and selection, and genetically transformed isolates obtained from the Methylobacterium strain.

As used herein, the term “emulsion” refers to a colloidal mixture of two immiscible liquids wherein one liquid is the continuous phase and the other liquid is the dispersed phase. In certain embodiments, the continuous phase is an aqueous liquid and the dispersed phase is liquid that is not miscible, or partially miscible, in the aqueous liquid.

As used herein, the phrase “essentially free of contaminating microorganisms” refers to a culture, fermentation broth, fermentation product, or composition where at least about 95% of the microorganisms present by amount or type in the culture, fermentation broth, fermentation product, or composition are the desired Methylobacterium or other desired microorganisms of pre-determined identity.

As used herein, the phrase “inanimate solid substance” refers to a substance which is insoluble or partially soluble in water or aqueous solutions and which is either non-living or which is not a part of a still-living organism from which it was derived.

As used herein, the phrase “mono-culture of Methylobacterium” refers to a Methylobacterium culture consisting of a single strain of Methylobacterium.

As used herein, the term “peptide” refers to any polypeptide of 50 amino acid residues or less.

As used herein, the term “tomato” refers to any Solanum lycopersicon hybrid or variety having either a determinant or indeterminant growth habit.

As used herein, the phrase “tomato seedlings” includes tomato plants from the germination stage through all vegetative stages.

As used herein, the phrase “tomato plants” includes tomato seedlings from the germination stage through all vegetative stages and tomato plants in all reproductive stages.

As used herein, the phrase “tomato plant” is inclusive of both tomato seedlings and tomato plants in all reproductive stages.

As used herein, the term “protein” refers to any polypeptide having 51 or more amino acid residues.

As used herein, a “pesticide” refers to an agent that is insecticidal, fungicidal, nematocidal, bacteriocidal, or any combination thereof.

As used herein, the phrase “bacteriostatic agent” refers to agents that inhibit growth of bacteria but do not kill the bacteria.

As used herein, the phrase “pesticide does not substantially inhibit growth of said Methylobacterium” refers to any pesticide that when provided in a composition comprising a fermentation product comprising a solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto, results in no more than a 50% inhibition of Methylobacterium growth when the composition is applied to a plant or plant part in comparison to a composition lacking the pesticide. In certain embodiments, the pesticide results in no more than a 40%, 20%, 10%, 5%, or 1% inhibition of Methylobacterium growth when the composition is applied to a plant or plant part in comparison to a composition lacking the pesticide.

As used herein, the term “PPFM bacteria” refers without limitation to bacterial species in the genus Methylobacterium other than M. nodulans.

As used herein, the phrase “solid substance” refers to a substance which is insoluble or partially soluble in water or aqueous solutions.

As used herein, the phrase “solid phase that can be suspended therein” refers to a solid substance that can be distributed throughout a liquid by agitation.

As used herein, the term “non-regenerable” refers to either a plant part or processed plant product that cannot be regenerated into a whole plant.

As used herein, the phrase “substantially all of the solid phase is suspended in the liquid phase” refers to media wherein at least 95%, 98%, or 99% of solid substance(s) comprising the solid phase are distributed throughout the liquid by agitation.

As used herein, the phrase “substantially all of the solid phase is not suspended in the liquid phase” refers to media where less than 5%, 2%, or 1% of the solid is in a particulate form that is distributed throughout the media by agitation.

As used herein, the phrase “resident microorganism” refers to resident bacteria, fungi or yeast.

As used herein, the phrase “substance that promotes growth of resident microorganisms on a plant or seed” refers to a carbon source, a nitrogen source, a phosphorous source, and combinations thereof.

To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

Methylobacterium-Containing Compositions Depleted of Substances that Promote Growth of Resident Bacteria on a Plant or Seed, Methods of their Use, and Methods of Making

Compositions comprising Methylobacterium that are depleted of substances that promote growth of resident bacteria on a plant or seed, methods of using the compositions to improve tomato production, and methods of making the compositions are provided herein. In certain embodiments of any of the aforementioned compositions, the composition comprises a solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto. In certain embodiments where the Methylobacterium is adhered to a solid substance, the composition comprises a colloid formed by the solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto and a liquid. In certain embodiments, the colloid is a gel. In certain embodiments of certain aforementioned compositions, composition is an emulsion that does not contain a solid substance.

Compositions that comprise a solid substance with adherent Methylobacterium grown thereon is provided. In certain embodiments, the adherent Methylobacterium can be at a titer of at least about 5×10⁸ CFU/gm to at least about 5×10¹³ CFU/gm or about 1×10¹⁴ CFU/gm and the composition is depleted of substances that promote growth of resident microorganisms on a plant or seed.

In certain embodiments, the compositions containing Methylobacterium provided or used herein are depleted of substances that promote growth of the resident microorganisms when one or more of those substances are absent or are essentially absent. In certain embodiments, the composition is depleted of substances that promote growth of the resident microorganisms when those substances are present at a percentage of no more than about 5%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total mass, mass/total volume, or total volume of the composition. In certain embodiments, substance that promotes growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorous source, a sulfur source, a magnesium source, and combinations thereof. Carbon sources include, but are not limited to, alcohols, monosaccharides, disaccharides, polysaccharides, lipids, fatty acids, and the like. Alcohols that are depleted include, but are not limited to, methanol, ethanol, glycerol, and the like. Nitrogen sources include, but are not limited to, ammonia and various compounds containing amino groups that can be metabolized by microorganisms. In certain embodiments, the substance that is depleted is a source of two or more of a carbon source, a nitrogen source, a phosphorous source, a sulfur source, and a magnesium source. For example, the composition that is depleted of amino acids or peptides and lacks other carbon or nitrogen sources is depleted for both a carbon and a nitrogen source. In certain embodiments, the composition comprises an agriculturally acceptable adjuvant and/or excipient.

Resident microorganisms on the plant or seed include, but are not limited to bacteria, fungi, and yeast. Substances that promote the growth of such microorganisms can be identified by methods including, but not limited to, assaying the plant or seed surface for the amount or number of microorganisms present prior to exposure of the plant or seed to the substance (or to a composition containing the substance), exposing the assayed plant or seed to the substance or composition in parallel with a control composition lacking the substance, and then re-assaying the plant or seed surface for the amount or number of microorganisms present after a suitable time interval and under suitable conditions of temperature to allow growth of the resident microorganisms. Assays for numbers of microorganisms include, but are not limited to, determinations of colony forming units per an amount of plant or seed exposed to the substance and the control.

Without seeking to be limited by theory, it is believed that the compositions containing Methylobacterium provided or used herein that are depleted of substances that promote growth of the resident microorganisms can result in superior results in comparison to other compositions containing such substances when applied to plants, plant parts, or seeds. Such superior results are believed to include, but are not limited to, improved plant yield, pathogen resistance, insect resistance, fruit ripening and the like. While not seeking to be limited by theory, it is believed that the compositions containing Methylobacterium that are depleted of substances that promote growth of the resident microorganisms allow for more efficient and or extensive colonization of the plant, part thereof, or seed as competition for one or more of space or nutrients by the resident microorganisms is reduced.

Also provided herein are methods for improving tomato production that comprise applying any of the aforementioned compositions or Methylobacterium provided herein to a tomato plant, tomato plant part, or tomato seed, and, optionally, growing the plant and/or harvesting seedlings, rootstock, scions, fruit, or seed from the plant or a plant grown from the seed. In certain embodiments, the composition coats or partially coats the tomato plant, plant part, or seed. The treated tomato plant or plant grown from the seed exhibits an increased rate of seedling growth, increased rate of root growth, an increased rate of leaf growth, increased seed production, a decreased cycle time (from seed planting to seed, rootstock, scion, or fruit production) and/or increased total biomass compared to an untreated control tomato plant or control tomato plant grown from untreated seed, thereby obtaining improved tomato production. In certain embodiments, application of the composition provides for at least about a 5%, 10%, 15%, 20%, 30% or 40% increase in root growth rate, leaf growth rate, seed, rootstock, scion, or fruit production rate, and/or increased total biomass in the tomato plant, tomato plant part, or a tomato plant derived therefrom in comparison to an untreated control tomato plant or control tomato plant grown from an untreated seed. In certain embodiments, application of the composition provides for about a 5% or 10% to about a 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, or 70% increase in root growth rate, leaf growth rate, seedling growth rate, seed production, fruit production, and/or increased total biomass in the plant, plant part, or a plant derived therefrom in comparison to an untreated control tomato plant or control tomato plant grown from an untreated seed. In certain embodiments, application of the composition provides for at least about a 5%, 10%, 15%, 20%, 30% or 40% decrease in cycle time (i.e. time from seed to progeny seed, to usable rootstock, to usable scion, graft, or fruit) in the treated tomato plant or a tomato plant grown from a treated seed in comparison to the untreated control tomato plant or control tomato plant grown from an un-treated seed. In certain embodiments, application of the composition provides for about a 5% or 10% to about a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% decrease in cycle time in the treated tomato plant or a tomato plant grown from a treated seed in comparison to an untreated control tomato plant or control tomato plant grown from an untreated seed. In certain embodiments, the tomato plant part is a leaf, a stem, a flower, a root, a tuber, or a seed. In certain embodiments, the method further comprises the steps of growing the plant and/or the step of harvesting at least one plant part selected from the group consisting of a leaf, a stem, a flower, a root, a fruit, or a seed from the tomato plant or plant part. In certain embodiments of any of the aforementioned methods, the methods further comprise obtaining a processed food or feed composition from the plant or plant part. In certain embodiments, the processed food composition comprises chopped or cut tomato fruit.

Also provided are methods of making a tomato plant or tomato plant seed treatment composition that comprises Methylobacterium and is depleted of substances that promote growth of resident bacteria on a plant or seed is provided herein. Such method comprises (i) growing a mono-culture or co-culture of Methylobacterium in media that comprises an aqueous phase, a liquid phase and a solid phase, or an emulsion, thereby obtaining a Methylobacterium-containing media; (ii) separating the Methylobacterium from at least one other portion of the Methylobacterium-containing media; and (iii) reconstituting the Methylobacterium in a matrix lacking substances that promote growth of resident bacteria on a plant or seed. In certain embodiments, the separation step is effected by centrifugation, filtration, or settling of the Methylobacterium-containing media and removal of excess liquid or emulsion therefrom. In certain embodiments, the substance that promotes growth of resident bacteria on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorous source, and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an agriculturally acceptable adjuvant and/or excipient. Still in certain embodiments; the Methylobacterium are grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown thereon. The solid substance is separated from the liquid phase of the Methylobacterium-containing media, and the solid substance with adherent Methylobacterium grown thereon is reconstituted in the aforementioned matrix. In certain embodiments of the methods, the Methylobacterium sp., is selected from the group consisting of M. aminovorans, M. extorquens, M. fujisawaense, M mesophilicum, M. radiotolerans, M rhodesianum, M. nodulans, M phyllosphaerae, M thiocyanatum, and M. oryzae. In certain embodiments of the methods, the Methylobacterium is not M. radiotolerans or M oryzae. In certain embodiments of the methods, the Methylobacterium is adhered to a solid substance. In certain embodiments of the methods, the Methylobacterium is adhered to the solid substance is combined with a liquid to form a composition that is a colloid. In certain embodiments of the methods, the colloid is a gel. In certain embodiments of the methods, the Methylobacterium adhered to the solid substance is provided by culturing the Methylobacterium in the presence of the solid substance. In certain embodiments of the methods, the composition comprises an emulsion. In certain embodiments of the methods, the Methylobacterium is provided by culturing the Methylobacterium in an emulsion.

Methods where Methylobacterium are cultured in biphasic media comprising a liquid phase and a solid substance have been found to significantly increase the resultant yield of Methylobacterium relative to methods where the Methylobacterium are cultured in liquid media alone. In certain embodiments, the methods can comprise growing the Methylobacterium in liquid media with a particulate solid substance that can be suspended in the liquid by agitation under conditions that provide for Methylobacterium growth. In certain embodiments where particulate solid substances are used, at least substantially all of the solid phase can thus be suspended in the liquid phase upon agitation. Such particulate solid substances can comprise materials that are about 1 millimeter or less in length or diameter. In certain embodiments, the degree of agitation is sufficient to provide for uniform distribution of the particulate solid substance in the liquid phase and/or optimal levels of culture aeration. However, in other embodiments provided herein, at least substantially all of the solid phase is not suspended in the liquid phase, or portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase. Non-particulate solid substances can be used in certain biphasic media where the solid phase is not suspended in the liquid phase. Such non-particulate solid substances include, but are not limited to, materials that are greater than about 1 millimeter in length or diameter. Such particulate and non-particulate solid substances also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to provide for increased surface areas for adherent growth of the Methylobacterium. Biphasic media where portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase can comprise a mixture of particulate and non-particulate solid substances. Such particulate and non-particulate solid substances used in any of the aforementioned biphasic media also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to provide for increased surface areas for adherent growth of the Methylobacterium. In certain embodiments, the media comprises a colloid formed by a solid and a liquid phase. A colloid comprising a solid and a liquid can be pre-formed and added to liquid media or can be formed in media containing a solid and a liquid. Colloids comprising a solid and a liquid can be formed by subjecting certain solid substances to a chemical and/or thermal change. In certain embodiments, the colloid is a gel. In certain embodiments, the liquid phase of the media is an emulsion. In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible, or only partially miscible, in the aqueous liquid. Liquids that are not miscible, or only partially miscible, in water include, but are not limited to, any of the following: (1) liquids having a miscibility in water that is equal to or less than that of pentanol, hexanol, or heptanol at 25 degrees C.; (2) liquids comprising an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid, or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols containing at least 5 carbons and sterols; (4) an animal oil, microbial oil, synthetic oil, plant oil, or combination thereof; and/or, (5) a plant oil is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower, and combinations thereof. In certain embodiments, the immiscible or partially immiscible liquid can comprises at least about 0.02% to about 20% of the liquid phase by mass. In certain embodiments, the methods can comprise obtaining a biphasic culture media comprising the liquid, the solid, and Methylobacterium and incubating the culture under conditions that provide for growth of the Methylobacterium. Biphasic culture medias comprising the liquid, the solid, and Methylobacterium can be obtained by a variety of methods that include, but are not limited to, any of: (a) inoculating a biphasic media comprising the liquid and the solid substance with Methylobacterium; (b) inoculating the solid substance with Methylobacterium and then introducing the solid substance comprising the Methylobacterium into the liquid media; (c) inoculating the solid substance with Methylobacterium, incubating the Methylobacterium on the solid substance, and then introducing the solid substance comprising the Methylobacterium into the liquid media; or (d) any combination of (a), (b), or (c). Methods and compositions for growing Methylobacterium in biphasic media comprising a liquid and a solid are disclosed in co-assigned U.S. patent application Ser. No. 13/907,161, filed May 31, 2013, which is incorporated herein by reference in its entirety, and in co-assigned International Patent Application PCT/US13/43722, filed May 31, 2013, which is incorporated herein by reference in its entirety.

Methods where Methylobacterium are cultured in media comprising an emulsion have also been found to significantly increase the resultant yield of Methylobacterium relative to methods where the Methylobacterium are cultured in liquid media alone. In certain embodiments, the methods for making the compositions provided herein can comprise growing the Methylobacterium agent in an emulsion under conditions that provide for Methylobacterium growth. Medias comprising the emulsion and Methylobacterium can be obtained by a variety of methods that include, but are not limited to, any of: (a) inoculating a media comprising the emulsion with Methylobacterium; (b) inoculating the aqueous liquid with the Methylobacterium, introducing the non-aqueous liquid, and mixing to form an emulsion; (c) inoculating the aqueous liquid with the Methylobacterium, introducing the non-aqueous liquid, and mixing to form an emulsion; or (d) any combination of (a), (b), or (c). In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible, or only partially miscible, in the aqueous liquid. Non-aqueous liquids that are not miscible, or only partially miscible, in water include, but are not limited to, any of the following: (1) liquids having a miscibility in water that is equal to or less than that of n-pentanol, n-hexanol, or n-heptanol at 25 degrees C.; (2) liquids comprising an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid, or any combination thereof; (3) alcohols is selected from the group consisting of aliphatic alcohols containing at least 5, 6, or 7 carbons and sterols; (4) an animal oil, microbial oil, synthetic oil, plant oil, or combination thereof; and/or, (5) a plant oil is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower, and combinations thereof. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid can comprise at least about 0.02% to about 20% of the emulsion by mass. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid can comprise at least about any of about 0.05%, 0.1%, 0.5%, or 1% to about 3%, 5%, 10%, or 20% of the emulsion by mass. Methods and compositions for growing Methylobacterium in media comprising an emulsion are disclosed in co-assigned U.S. Provisional Patent Application No. 61/829,987, filed May 31, 2013, which is incorporated herein by reference in its entirety.

In certain embodiments, the fermentation broth, fermentation broth product, or compositions that comprise Methylobacterium sp. can further comprise one or more introduced microorganisms of pre-determined identity other than Methylobacterium. Other microorganisms that can be added include, but are not limited to, microorganisms that are biopesticidal or provide some other benefit when applied to a plant or plant part. Biopesticidal or otherwise beneficial microorganisms thus include, but are not limited to, various Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp., and Trichoderma sp. Microbial biopesticides can be a bacterium, fungus, virus, or protozoan. Particularly useful biopesticidal microorganisms include various Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma vixens, and Streptomyces lydicus strains. Other microorganisms that are added can be genetically engineered or naturally occurring isolates that are available as pure cultures. In certain embodiments, it is anticipated that the bacterial or fungal microorganism can be provided in the fermentation broth, fermentation broth product, or composition in the form of a spore.

In certain embodiments, the liquid culture medium is prepared from inexpensive and readily available components, including, but not limited to, inorganic salts such as potassium phosphate, magnesium sulfate and the like, carbon sources such as glycerol, methanol, glutamic acid, aspartic acid, succinic acid and the like, and amino acid blends such as peptone, tryptone, and the like. Exemplary liquid media that can be used include, but are not limited to, ammonium mineral salts (AMS) medium (Whittenbury et al., 1970), Vogel-Bonner (VB) minimal culture medium (Vogel and Bonner, 1956), and LB broth (“Luria—Bertani Broth”).

In general, the solid substance used in the methods and compositions that provide for the efficient growth of Methylobacterium can be any suitable solid substance which is insoluble or only partially soluble in water or aqueous solutions. Such suitable solid substances are also non-bacteriocidal or non-bacteriostatic with respect to Methylobacterium when the solid substances are provided in the liquid culture media. In certain embodiments, such suitable solid substances are also solid substances that are readily obtained in sterile form or rendered sterile. Solid substances used herein can be sterilized by any method that provides for removal of contaminating microorganisms and thus include, but are not limited to, methods such as autoclaving, irradiation, chemical treatment, and any combination thereof. These solid substances include natural substances of animal, plant, microbial, fungal, or mineral origin, manmade substances, or combinations of natural and manmade substances. In certain embodiments, the solid substances are inanimate solid substances. Inanimate solid substances of animal, plant, microbial, or fungal origin can be obtained from animals, plants, microbes, or fungi that are unviable (i.e. no longer living) or that have been rendered unviable. Diatom shells are thus inanimate solid substances when previously associated diatom algae have been removed or otherwise rendered inviable. Since diatom shells are inanimate solid substances, they are not considered to be photosynthetic organisms or photosynthetic microorganisms. In certain embodiments, solid substances include, but are not limited to, sand, silt, soil, clay, ash, charcoal, diatomaceous earth and other similar minerals, ground glass or glass beads, ground ceramic materials, ceramic beads, bentonite, kaolin, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite, and combinations thereof. In certain embodiments, the solid substance can be a polymer or polymeric beads. Polymers that can be used as a solid substance include, but are not limited to, various polysaccharides such as cellulosic polymers and chitinous polymers which are insoluble or only partially soluble in water or aqueous solutions, agar (i.e. galactans), and combinations thereof. In certain embodiments, the solid substance can be an insoluble or only partially soluble salt crystal. Salt crystals that can be used include, but are not limited to, insoluble or only partially soluble carbonates, chromates, sulfites, phosphates, hydroxides, oxides, and sulfides. In certain embodiments, the solid substance can be a microbial cell, fungal cell, microbial spore, or fungal spore. In certain embodiments, the solid substance can be a microbial cell or microbial spore wherein the microbial cell or microbial spore is not a photosynthetic microorganism. In certain embodiments, the microbial cell or microbial spore is not a photosynthetic microorganism, where the photosynthetic microorganism is selected from the group consisting of algae, cyanobacteria, diatoms, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis camerae, Sargassum, and Ulva. In still other embodiments, the solid substance can be an inactivated (i.e. unviable) microbial cell, fungal cell, microbial spore, or fungal spore. In still other embodiments, the solid substance can be a quiescent (i.e. viable but not actively dividing) microbial cell, fungal cell, microbial spore, or fungal spore. In still other embodiments, the solid substance can be cellular debris of microbial origin. In still other embodiments, the solid substance can be particulate matter from any part of a plant. Plant parts that can be used to obtain the solid substance include, but are not limited to, cobs, husks, hulls, leaves, roots, flowers, stems, barks, seeds, and combinations thereof. Products obtained from processed plant parts including, but not limited to, bagasse, wheat bran, soy grits, crushed seed cake, stover, and the like can also be used. Such plant parts, processed plants, and/or processed plant parts can be milled to obtain the solid material in a particulate form that can be used. In certain embodiments, wood or a wood product including, but not limited to, wood pulp, sawdust, shavings, and the like can be used. In certain embodiments, the solid substance can be a particulate matter from an animal(s), including, but not limited to, bone meal, gelatin, ground or powdered shells, hair, macerated hide, and the like.

In certain embodiments, the solid substance is provided in a particulate form that provides for distribution of the solid substance in the culture media. In certain embodiments, the solid substance is comprised of particle of about 2 microns to about 1000 microns in average length or average diameter. In certain embodiments, the solid substance is comprised of particle of about 1 microns to about 1000 microns in average length or average diameter. In certain embodiments, the solid substance is a particle of about 1, 2, 4, 10, 20, or 40 microns to any of about 100, 200, 500, 750, or 1000 microns in average length or average diameter. Desirable characteristics of particles used in the methods and compositions provided herein include suitable wettability such that the particles can be suspended throughout the media upon agitation.

In certain embodiments, the solid substance is provided in the media as a colloid wherein the continuous phase is a liquid and the dispersed phase is the solid. Suitable solids that can be used to form colloids in liquid media used to grow Methylobacterium include, but are not limited to, various solids that are referred to as hydrocolloids. Such hydrocolloids used in the media, methods and compositions provided herein can be hydrophilic polymers, of plant, animal, microbial, or synthetic origin. Hydrocolloid polymers used in the methods can contain many hydroxyl groups and/or can be polyelectrolytes. Hydrocolloid polymers used in the compositions and methods provided herein include, but are not limited to, agar, alginate, arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin, gellan, β-glucan, guar gum, gum arabic, locust bean gum, pectin, starch, xanthan gum, and mixtures thereof. In certain embodiments, the colloid used in the media, methods, and compositions provided herein can comprise a hydrocolloid polymer and one or more proteins.

In certain embodiments, the solid substance can be a solid substance that provides for adherent growth of Methylobacterium on the solid substance. Methylobacterium that are adhered to a solid substance are Methylobacterium that cannot be substantially removed by simply washing the solid substance with the adherent Methylobacterium with growth media whereas non-adherent Methylobacterium can be substantially removed by washing the solid substance with liquid growth media. In this context, “substantially removed” means that at least about 30%, 40%, 50%, 60%, 70%, or 80% the Methylobacterium present are removed when the solid substance is washed with three volumes of liquid growth media. Such washing can be effected by a variety of methods including, but not limited to, decanting liquid from a washed solid phase or passing liquid through a solid phase on a filter that permits flow through of bacteria in the liquid. In certain embodiments, the adherent Methylobacterium that are associated with the solid can include both Methylobacterium that are directly attached to the solid and/or Methylobacterium that are indirectly attached to the solid substance. Methylobacterium that are indirectly attached to the solid substance include, but are not limited to, Methylobacterium that are attached to another Methylobacterium or to another microorganism that is attached to the solid substance, Methylobacterium that are attached to the solid substance by being attached to another substance that is attached to the solid substance, and the like. In certain embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the Methylobacterium in the fermentation broth, fermentation broth product, or compositions are Methylobacterium that are adhered to the solid substance. In certain embodiments, adherent Methylobacterium can be present on the surface of the solid substance in the fermentation broth, fermentation broth product, or composition at a density of at least about 1 Methylobacterium/20 square micrometers, of at least about 1 Methylobacterium/10 square micrometers, of at least about 1 Methylobacterium/10 square micrometers, of at least about 1 Methylobacterium/5 square micrometers, of at least about 1 Methylobacterium/2 square micrometers, or of at least about 1 Methylobacterium/square micrometer. In certain embodiments, adherent Methylobacterium can be present on the surface of the solid substance in the fermentation broth, fermentation broth product, or composition at a density of at least about 1 Methylobacterium/20 square micrometers to about 1 Methylobacterium/square micrometer, of at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/square micrometer, of at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/square micrometer, of at least about 1 Methylobacterium/5 square micrometers to about 1 Methylobacterium/square micrometer, or of at least about 1 Methylobacterium/2 square micrometers to about 1 Methylobacterium/square micrometer. In certain embodiments, adherent Methylobacterium can be present on the surface of the solid substance in the fermentation broth, fermentation broth product, or composition at a density of at least about 1 Methylobacterium/20 square micrometers to about 1 Methylobacterium/2 square micrometers, of at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/2 square micrometers, of at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/2 square micrometers, or of at least about 1 Methylobacterium/5 square micrometers to about 1 Methylobacterium/2 square micrometers. Biphasic fermentation broths provided herein can comprise a liquid phase that contains non-adherent Methylobacterium. In certain embodiments, titers of non-adherent Methylobacterium in the liquid phase can be less than about 100,000, 10,000, or 1,000 CFU/ml.

Biphasic culture methods provided can yield fermentation broths with Methylobacterium at a titer of greater than about 5×10⁸ colony-forming units per milliliter, at a titer of greater than about 1×10⁹ colony-forming units per milliliter, at a titer of greater than about 1×10¹⁰ colony-forming units per milliliter, at a titer of at least about 3×10¹⁰ colony-forming units per milliliter. In certain embodiments, fermentation broths provided herein can comprise Methylobacterium at a titer of at least about 5×10⁸ colony-forming units per milliliter to at least about 3×10¹⁰ colony-forming units per milliliter, at least about 5×10⁸ colony-forming units per milliliter to at least about 4×10¹⁰ colony-forming units per milliliter, or at least about 5×10⁸ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter. In certain embodiments, fermentation broths provided herein can comprise Methylobacterium at a titer of at least about 1×10⁹ colony-forming units per milliliter to at least about 3×10¹⁰ colony-forming units per milliliter, at least about 1×10⁹ colony-forming units per milliliter to at least about 4×10¹⁰ colony-forming units per milliliter, or at least about 1×10⁹ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter. In certain embodiments, fermentation broths provided herein will comprise Methylobacterium at a titer of at least about 1×10¹⁰ colony-forming units per milliliter to at least about 3×10¹⁰ colony-forming units per milliliter, at least about 1×10¹⁰ colony-forming units per milliliter to at least about 4×10¹⁰ colony-forming units per milliliter, or at least about 1×10¹⁰ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter. In certain embodiments, fermentation broths provided herein will comprise Methylobacterium at a titer of, at least about 3×10¹⁰ colony-forming units per milliliter to at least about 4×10¹⁰ colony-forming units per milliliter, or at least about 3×10¹⁰ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter.

Solid substances with adherent Methylobacterium can be obtained as fermentation products can be used to make various compositions useful for treating plants or plant parts to improve plant yield, plant insect resistance, plant fungal disease resistance, and/or to improve tomato production. In certain embodiments, the composition comprises Methylobacterium and is depleted of substances that promote growth of resident bacteria. Compositions provided herein comprising Methylobacterium, solid substances with Methylobacterium grown thereon, or comprising emulsions with Methylobacterium grown therein can be used to treat plants or plant parts. Plants, plant parts, and, in particular, plant seeds that have been at least partially coated or coated with the fermentation broth products or compositions comprising Methylobacterium are thus provided. Also provided are processed plant products that contain the fermentation broth products or compositions with Methylobacterium or adherent Methylobacterium. Solid substances with adherent Methylobacterium can be used to make various compositions that are particularly useful for treating plant seeds. Seeds that have been at least partially coated with the fermentation broth products or compositions are thus provided. Also provided are processed seed products, including, but not limited to, meal, flour, feed, and flakes that contain the fermentation broth products or compositions provided herein. In certain embodiments, the processed plant product will be non-regenerable (i.e. will be incapable of developing into a plant). In certain embodiments, the solid substance used in the fermentation product or composition that at least partially coats the plant, plant part, or plant seed or that is contained in the processed plant, plant part, or seed product comprises a solid substance and associated or adherent Methylobacterium that can be readily identified by comparing a treated and an untreated plant, plant part, plant seed, or processed product thereof. Partial coating of a plant, a plant part, or a seed includes, but is not limited to coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of the plant, plant part, or plant seed.

Methods of preparing a plant or plant seed treatment composition that comprises Methylobacterium and is depleted of substances that promote growth of resident bacteria on a plant or seed are also provided herein. Such methods can comprise (i) growing a mono-culture or co-culture of Methylobacterium in media that comprises: (a) an aqueous phase; (b) a liquid phase and a solid phase; or (c) an emulsion, thereby obtaining a Methylobacterium-containing media; (ii) separating the Methylobacterium from at least one other portion of the Methylobacterium-containing media; and (iii) reconstituting the Methylobacterium in a matrix lacking substances that promote growth of resident bacteria on a plant or seed. In certain embodiments, the separation step is effected by centrifugation, filtration, or settling of the Methylobacterium-containing media and removal of excess liquid or emulsion therefrom. In certain embodiments where the Methylobacterium are grown in the presence of a solid substance, the separation will provide a fraction containing Methylobacterium with adherent growth to the solid substance and some non-adherent Methylobacterium that can be reconstituted in the matrix. In certain embodiments, the substance that promotes growth of resident bacteria on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorous source, a sulfur source, a magnesium source, and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an agriculturally acceptable adjuvant and/or excipient. In certain embodiments; the Methylobacterium are grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown thereon. The solid substance is separated from the liquid phase of the Methylobacterium-containing media, and the solid substance with adherent Methylobacterium grown thereon is reconstituted in the aforementioned matrix. In certain embodiments, the matrix can be a liquid including, but not limited to, water, and aqueous buffer depleted of substances that promote growth of resident bacteria on a plant or seed, or an aqueous solution depleted of substances that promote growth of resident bacteria on a plant or seed.

In certain embodiments, the Methylobacterium sp. that improve tomato production can be identified by testing newly isolated candidate Methylobacterium sp. for the presence of polymorphic nucleic acid sequences that are present in exemplary Methylobacterium sp. provided herein that improve tomato seedling growth rates and that are absent from Methylobacterium sp. that do not improve tomato seedling growth rates. In certain embodiments, the polymorphic nucleic acid sequences that are present in the identified Methylobacterium sp. that improves tomato production are also present in one or more of the exemplary Methylobacterium sp. isolates NLS0037 provided herein that improves tomato seedling growth rate but are absent from one or more of the Methylobacterium sp. isolates that do not improve tomato seedling growth rates. Such nucleic acid polymorphisms that occur in the Methylobacterium sp. that improve tomato production can include, but are not limited to, single nucleotide polymorphisms, RFLP, AFLP and/or other DNA variations such as repetitive sequences, insertion sequences, transposons, and genomic islands occurring as a result of insertions, deletions, and substitutions (Indels) in the bacterial genome which includes both the chromosomal DNA as well as any extrachromosomal nucleic acid elements that can be present in the Methylobacterium sp. that improve tomato production. Such extrachromosomal nucleic acid elements include, but are not limited to, plasmids, bacteriophage DNA or RNA, and the like. Methods used to identify such nucleotide polymorphisms include, but are not limited to, single base extension (SBE) techniques, allele specific hybridization (ASH), real-time PCR detection (e.g. TaqMan™; U.S. Pat. Nos. 5,804,375; 5,538,848; 5,487,972; and 5,210,015, which are each incorporated herein by reference in their entireties), combinations of ASH and RT-PCR (KASP™ detection systems, LGC Genomics, Middlesex, UK) and deep sequencing techniques (U.S. Patent Appl. No. 20120264632, incorporated herein by reference in its entirety).

Also provided herein are compositions, methods of making the compositions, and methods of using the compositions to improve tomato production. Such improved tomato production includes, but is not limited to, increased root growth rate, leaf growth rate, seedling growth rate, seed production, fruit production, scion production, rootstock production, and/or increased total biomass in comparison to an untreated control tomato plant. In certain embodiments, the compositions or methods comprise or use any of the following Methylobacterium sp. isolates provided in the following Table 1 or derivatives of the isolates. In certain embodiments, such derivatives can include variants but are not limited to, variants of the isolates obtained by selection, variants of the isolates selected by mutagenesis and selection, and genetically transformed isolates obtained from the isolates.

TABLE 1 Methylobacterium sp. isolates ISOLATE NLS USDA ARS No. No. NRRL No.¹ ISO01 NLS0046 NRRL B-50929 ISO02 NLS0020 NRRL B-50930 ISO03 NLS0017 NRRL B-50931 ISO04 NLS0042 NRRL B-50932 ISO05 NLS0089 NRRL B-50933 ISO06 NLS0068 NRRL B-50934 ISO07 NLS0065 NRRL B-50935 ISO08 NLS0069 NRRL B-50936 ISO09 NLS0062 NRRL B-50937 ISO10 NLS0064 NRRL B-50938 ISO11 NLS0021 NRRL B-50939 ISO12 NLS0066 NRRL B-50940 ISO13 NLS0037 NRRL B-50941 ISO14 NLS0038 NRRL B-50942 ¹Deposit number for strain to be deposited with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) of the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A. under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Subject to 37 CFR §1.808(b), all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of any patent from this patent application.

Co-assigned patent applications that disclose additional specific uses of the Methylobacterium strains of Table 1 such as: (1) increasing corn yield (U.S. 61/911,780, filed Dec. 4, 2013; and International application claiming benefit of the same filed on Dec. 4, 2014); (2) increasing soybean yield (U.S. 61/911,698, filed Dec. 4, 2013; and International application claiming benefit of the same filed on Dec. 4, 2014); (3) improving lettuce cultivation (International Patent Application PCT/US14/68558 filed on Dec. 4, 2014); (4) providing fungal disease resistance (U.S. 62/045,950, filed Sep. 4, 2014; U.S. 62/013,464, filed Jun. 17, 2014) and are each incorporated herein by reference in their entireties. Specifically incorporated herein by reference in their entireties are the genomic nucleic acid sequences of NLS017, NLS020, NLS037, NLS042, NLS065, and NLS066 that are disclosed in International application filed on Dec. 4, 2014 and claiming benefit of U.S. 61/954,840, filed Mar. 18, 2014, and U.S. 61/911,516, filed Dec. 4, 2013. Such genomic nucleic acid sequences can be used to identify compositions, plant parts, plant seeds, or processed plant products comprising NLS017, NLS020, NLS037, NLS042, NLS065, and NLS066.

Also provided herein are Methylobacterium sp. that provide for improved tomato production where the Methylobacterium sp. have any of: (i) at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594; or (ii) at least one gene encoding at least one protein that is orthologous to a reference protein of Table 4. A Methylobacterium sp. has at least one gene that is orthologous to a protein having an amino acid sequence of at least one of SEQ ID NO: 1-4594, or to the corresponding SEQ ID NO of a reference protein of Table 4, when a chromosome and/or any extrachromosomal DNA in that Methylobacterium sp. contains a gene encoding a protein that has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity across the entire length of the amino acid sequence of at least one of SEQ ID NO: 1-4594. The Methylobacterium sp. can also have at least two, three, four, six, eight, 10, 15, or 20 genes encoding proteins that are orthologous to proteins having an amino acid sequence of SEQ ID NO: 1-4594 or encoding proteins that are orthologous to the corresponding SEQ ID NO of a reference protein of Table 4. In certain embodiments, the Methylobacterium sp. can contain at least one gene encoding a protein that is orthologous to a reference protein having the amino acid sequence of SEQ ID NO: 1-2684 of Table 4. In certain embodiments, the Methylobacterium sp. can contain at least one gene encoding a protein that is orthologous to reference protein having the amino acid sequence of SEQ ID NO: 2585-4594 of Table 4. In certain embodiments, the Methylobacterium sp. can contain at least one gene encoding a protein that is orthologous to reference protein having the amino acid sequence of SEQ ID NO: 2969 or 212 of Table 4. Examples of proteins that are orthologous to SEQ ID NO: 2969 include, but are not limited to, the orthologous proteins identified as transcriptional regulator XRE family proteins of SEQ ID NO: 2969 and 399 that are provided in Table 4. Examples of proteins that are orthologous to SEQ ID NO: 212 include, but are not limited to, proteins having the amino acid sequence of SEQ ID NO: 212 and 2828 that are similar to proteins identified as members of the LysR family transcriptional regulators. Compositions comprising any of the aforementioned Methylobacterium sp. and an agriculturally acceptable excipient, adjuvant, or combination thereof are also provided along with tomato seeds or leaves that are at least partially coated with such compositions and methods of using such compositions as seed or foliar treatments to improve tomato production.

A Methylobacterium sp. can be determined to contain a gene encoding a protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 by a variety of different techniques. In certain embodiments, a Methylobacterium sp. can be determined to contain a gene encoding a protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 by assembling a complete electronic genomic sequence comprising chromosomal and extrachromosomal DNA sequences present in that Methylobacterium sp. with a computer and associated software, and determining if any of the open reading frames (ORF) present in that DNA sequence encode a protein having the aforementioned percent sequence identity. In such embodiments, the ORF can be identified by performing a six-way translation of the electronically assembled sequence and querying the translated with an amino acid sequence of SEQ ID NO: 1-4594 or the corresponding SEQ ID NO: of a reference protein of Table 4. In other embodiments, the present or absence of a given sequence within a Methylobacterium sp. an amino acid sequence of SEQ ID NO: 1-4594 or the corresponding SEQ ID NO: of a reference protein of Table 4 can be determined by a nucleic acid analysis or protein analysis technique. Examples of nucleic acid sequences that encode the proteins of SEQ ID NO:1-4594 include, but are not limited to, SEQ ID NO: 4595-9188, respectively. Such nucleic acid analyses include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore based detection, branched DNA analyses, combinations thereof, and the like. Protein analysis techniques include, but are not limited to, immuno-detection, mass spectroscopy, combinations thereof, and the like.

Compositions provided herein that are useful for treating tomato plants or plant parts that comprise Methylobacterium, and/or are depleted of substances that promote growth of resident bacteria on a plant or seed, contain a solid substance with adherent Methylobacterium grown thereon, or that comprise emulsions with Methylobacterium grown therein can also further comprise an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. An agriculturally acceptable adjuvant or an agriculturally acceptable excipient is typically an ingredient that does not cause undue phytotoxicity or other adverse effects when exposed to a plant or plant part. In certain embodiments, the solid substance can itself be an agriculturally acceptable adjuvant or an agriculturally acceptable excipient so long as it is not bacteriocidal or bacteriostatic to the Methylobacterium. In other embodiments, the composition further comprises at least one of an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. Any of the aforementioned compositions can also further comprise a pesticide. Pesticides used in the composition include, but are not limited to, an insecticide, a fungicide, a nematocide, and a bacteriocide. In certain embodiments, the pesticide used in the composition is a pesticide that does not substantially inhibit growth of the Methylobacterium. As Methylobacterium are gram negative bacteria, suitable bacteriocides used in the compositions can include, but are not limited to, bacteriocides that exhibit activity against gram positive bacteria but not gram negative bacteria. Compositions provided herein can also comprise a bacteriostatic agent that does not substantially inhibit growth of the Methylobacterium. Bacteriostatic agents suitable for use in compositions provided herein include, but are not limited to, those that exhibit activity against gram positive bacteria but not gram negative bacteria. Any of the aforementioned compositions can also be an essentially dry product (i.e. having about 5% or less water content), a mixture of the composition with an emulsion, or a suspension. Any of the compositions provided herein can be used to coat or partially coat a plant, plant, part, or plant seed. Partial coating of a plant, a plant part, or a seed includes, but is not limited to coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of the plant, plant part, or plant seed.

Agriculturally acceptable adjuvants used in the compositions that comprise Methylobacterium include, but are not limited to, components that enhance product efficacy and/or products that enhance ease of product application. Adjuvants that enhance product efficacy can include various wetters/spreaders that promote adhesion to and spreading of the composition on plant parts, stickers that promote adhesion to the plant part, penetrants that can promote contact of the active agent with interior tissues, extenders that increase the half-life of the active agent by inhibiting environmental degradation, and humectants that increase the density or drying time of sprayed compositions. Wetters/spreaders used in the compositions can include, but are not limited to, non-ionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, organo-silicate surfactants, and/or acidified surfactants. Stickers used in the compositions can include, but are not limited to, latex-based substances, terpene/pinolene, and pyrrolidone-based substances. Penetrants can include mineral oil, vegetable oil, esterified vegetable oil, organo-silicate surfactants, and acidified surfactants. Extenders used in the compositions can include, but are not limited to, ammonium sulphate, or menthene-based substances. Humectants used in the compositions can include, but are not limited to, glycerol, propylene glycol, and diethyl glycol. Adjuvants that improve ease of product application include, but are not limited to, acidifying/buffering agents, anti-foaming/de-foaming agents, compatibility agents, drift-reducing agents, dyes, and water conditioners. Anti-foaming/de-foaming agents used in the compositions can include, but are not limited to, dimethopolysiloxane. Compatibility agents used in the compositions can include, but are not limited to, ammonium sulphate. Drift-reducing agents used in the compositions can include, but are not limited to, polyacrylamides, and polysaccharides. Water conditioners used in the compositions can include, but are not limited to, ammonium sulphate.

Methods of treating plants and/or plant parts with the fermentation broths, fermentation broth products, and compositions comprising Methylobacterium are also provided herein. Treated plants, and treated plant parts obtained therefrom, include, but are not limited to, a tomato, plant. Plant parts that are treated include, but are not limited to, leaves, stems, flowers, roots, seeds, fruit, tubers, coleoptiles, and the like. Seeds or other propagules of any of the aforementioned plants can be treated with the fermentation broths, fermentation broth products, fermentation products, and/or compositions provided herein.

In certain embodiments, plants and/or plant parts are treated by applying the fermentation broths, fermentation broth products, fermentation products, and compositions that comprise Methylobacterium as a spray. Such spray applications include, but are not limited to, treatments of a single plant part or any combination of plant parts. Spraying can be achieved with any device that will distribute the fermentation broths, fermentation broth products, fermentation products, and compositions to the plant and/or plant part(s). Useful spray devices include a boom sprayer, a hand or backpack sprayer, crop dusters (e.g. aerial spraying), and the like. Spraying devices and or methods providing for application of the fermentation broths, fermentation broth products, fermentation products, and compositions to either one or both of the adaxial surface and/or abaxial surface can also be used. Plants and/or plant parts that are at least partially coated with any of a biphasic fermentation broth, a fermentation broth product, fermentation product, or compositions that comprise a solid substance with Methylobacterium adhered thereto are also provided herein. Also provided herein are processed plant products that comprise a solid substance with Methylobacterium adhered thereto. Any of the compositions provided herein can be used to coat or partially coat a plant, plant, part, or plant seed. Partial coating of a plant, a plant part, or a seed includes, but is not limited to coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of the plant, plant part, or plant seed.

In certain embodiments, tomato seeds are treated by exposing the seeds to the fermentation broths, fermentation broth products, fermentation products, and compositions that comprise Methylobacterium. Seeds can be treated with the fermentation broths, fermentation broth products, and compositions provided herein by methods including, but not limited to, imbibition, coating, spraying, and the like. In certain embodiments, surface sterilized seeds are treated with a composition comprising Methylobacterium. In certain embodiments, non-sterilized seeds (i.e. seeds that have not been subjected to surface sterilization) are treated with a composition comprising Methylobacterium that has been depleted of substances that promote growth of resident microorganisms on the seed. Seed treatments can be effected with both continuous and/or a batch seed treaters. In certain embodiments, the coated seeds may be prepared by slurrying seeds with a coating composition containing a fermentation broth, fermentation broth product, or compositions that comprise the solid substance with Methylobacterium and air drying the resulting product. Air drying can be accomplished at any temperature that is not deleterious to the seed or the Methylobacterium, but will typically not be greater than 30 degrees Centigrade. The proportion of coating that comprises a solid substance and Methylobacterium includes, but is not limited to, a range of 0.1 to 25% by weight of the seed, 0.5 to 5% by weight of the seed, and 0.5 to 2.5% by weight of seed. Partial coating of a seed can includes, but is not limited to coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of the seed. In certain embodiments, a solid substance used in the seed coating or treatment will have Methylobacterium adhered thereon. In certain embodiments, a solid substance used in the seed coating or treatment will be associated with Methylobacterium and will be a fermentation broth, fermentation broth product, or composition obtained by the methods provided herein. Various seed treatment compositions and methods for seed treatment disclosed in U.S. Pat. Nos. 5,106,648, 5,512,069, and 8,181,388 are incorporated herein by reference in their entireties and can be adapted for use with an active agent comprising the fermentation broths, fermentation broth products, or compositions provided herein. In certain embodiments, the composition used to treat the seed can contain agriculturally acceptable excipients that include, but are not limited to, woodflours, clays, activated carbon, diatomaceous earth, fine-grain inorganic solids, calcium carbonate and the like. Clays and inorganic solids that can be used with the fermentation broths, fermentation broth products, or compositions provided herein include, but are not limited to, calcium bentonite, kaolin, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and mixtures thereof. Agriculturally acceptable adjuvants that promote sticking to the seed that can be used include, but are not limited to, polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes, latex polymers, celluloses including ethylcelluloses and methylcelluloses, hydroxy methylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, alginates, dextrins, malto-dextrins, polysaccharides, fats, oils, proteins, karaya gum, jaguar gum, tragacanth gum, polysaccharide gums, mucilage, gum arabics, shellacs, vinylidene chloride polymers and copolymers, soybean-based protein polymers and copolymers, lignosulfonates, acrylic copolymers, starches, polyvinylacrylates, zeins, gelatin, carboxymethylcellulose, chitosan, polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, alginate, ethylcellulose, polychloroprene and syrups or mixtures thereof. Other useful agriculturally acceptable adjuvants that can promote coating include, but are not limited to, polymers and copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer and water-soluble waxes. Various surfactants, dispersants, anticaking-agents, foam-control agents, and dyes disclosed herein and in U.S. Pat. No. 8,181,388 can be adapted for use with an active agent comprising the fermentation broths, fermentation broth products, or compositions provided herein.

In certain embodiments, PPFMs that are used to increase tomato production could also be applied in a hydroponic solution as an addition to the hydroponic pool. Such hydroponic solutions are solutions comprising at least minerals necessary for tomato plant growth. Hydroponic solutions suitable for growth of tomato plants and seedlings include, but are not limited, to those described in U.S. Pat. No. 8,091,275 and U.S. Pat. No. 7,818,916, which are each incorporated herein by reference in their entireties with respect to the hydroponic solutions disclosed therein.

Provided herein are compositions that comprise Methylobacterium that provide increased tomato fruit, scion, or rootstock production and increased tomato seedling growth relative to untreated plants that have not been exposed to the compositions. In certain embodiments, plant parts, including, but not limited to, a seed, a leaf, a fruit, a stem, a root, a tuber, or a coleoptile can be treated with the compositions provided herein to increase tomato production. Treatments or applications can include, but are not limited to, spraying, coating, partially coating, immersing, and/or imbibing the plant or plant parts with the compositions provided herein. In certain embodiments, a seed, a leaf, a fruit, a stem, a root, a tuber, or a coleoptile can be immersed and/or imbibed with a liquid, semi-liquid, emulsion, or slurry of a composition provided herein. Such seed immersion or imbibition can be sufficient to provide for improved tomato production in a treated plant or plant part in comparison to an untreated plant or plant part. Improved tomato production includes, but is not limited, to increased seedling growth, root growth, increased leaf growth, increased seed, scion, or rootstock production, and/or increased total biomass in comparison to untreated control plants. In certain embodiments, plant seeds can be immersed and/or imbibed for at least 1, 2, 3, 4, 5, or 6 hours. Such immersion and/or imbibition can, in certain embodiments, be conducted at temperatures that are not deleterious to the plant seed or the Methylobacterium. In certain embodiments, the seeds can be treated at about 15 to about 30 degrees Centigrade or at about 20 to about 25 degrees Centigrade. In certain embodiments, seed imbibition and/or immersion can be performed with gentle agitation.

Compositions provided herein comprising Methylobacterium are therefore expected to be useful in improving tomato production.

In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with Methylobacterium at a titer of at least about 1×10⁶ colony-forming units per milliliter, at least about 5×10⁶ colony-forming units per milliliter, at least about 1×10⁷ colony-forming units per milliliter, at least about 5×10⁸ colony-forming units per milliliter, at least about 1×10⁹ colony-forming units per milliliter, at least about 1×10¹⁰ colony-forming units per milliliter, or at least about 3×10¹⁰ colony-forming units per milliliter. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improving tomato production can be a composition with Methylobacterium at a titer of about least about 1×10⁶ colony-forming units per milliliter, at least about 5×10⁶ colony-forming units per milliliter, at least about 1×10⁷ colony-forming units per milliliter, or at least about 5×10⁸ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improving tomato production can be a fermentation broth product with a Methylobacterium titer of a solid phase of that product is at least about 5×10⁸ colony-forming units per milliliter to at least about 5×10¹³ colony-forming units of Methylobacterium per gram of the solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improving tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per gram, at least about 5×10⁶ colony-forming units per gram, at least about 1×10⁷ colony-forming units per gram, or at least about 5×10⁸ colony-forming units per gram to at least about 6×10¹⁰ colony-forming units of Methylobacterium per gram of particles in the composition containing the particles that comprise a solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improving tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per mL, at least about 5×10⁶ colony-forming units per mL, at least about 1×10⁷ colony-forming units per mL, or at least about 5×10⁸ colony-forming units per mL to at least about 6×10¹⁰ colony-forming units of Methylobacterium per mL in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium adhered to a solid substance is provided therein or grown therein. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improving tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per mL, at least about 5×10⁶ colony-forming units per mL, at least about 1×10⁷ colony-forming units per mL, or at least about 5×10⁸ colony-forming units per ml, to at least about 6×10¹⁰ colony-forming units of Methylobacterium per mL of in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium is provided therein or grown therein.

In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with a Methylobacterium sp. at a titer of at least about 1×10⁴ colony-forming units per milliliter, at least about 1×10⁵ colony-forming units per milliliter, at least about 1×10⁶ colony-forming units per milliliter, at least about 5×10⁶ colony-forming units per milliliter, at least about 1×10⁷ colony-forming units per milliliter, at least about 5×10⁸ colony-forming units per milliliter, at least about 1×10⁹ colony-forming units per milliliter, at least about 1×10¹⁰ colony-forming units per milliliter, or at least about 3×10¹⁰ colony-forming units per milliliter. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with Methylobacterium sp. at a titer of at least about 1×10⁴ colony-forming units per milliliter, at least about 1×10⁵ colony-forming units per milliliter, about least about 1×10⁶ colony-forming units per milliliter, at least about 5×10⁶ colony-forming units per milliliter, at least about 1×10⁷ colony-forming units per milliliter, or at least about 5×10⁸ colony-forming units per milliliter to at least about 6×10¹⁰ colony-forming units per milliliter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a fermentation broth product with a Methylobacterium sp. titer of a solid phase of that product is at least about 1×10⁴ colony-forming units per gram, at least about 1×10⁵ colony-forming units per gram, at least about 1×10⁶ colony-forming units per gram, at least about 5×10⁶ colony-forming units per gram, at least about 1×10⁷ colony-forming units per gram, at least about 5×10⁸ colony-forming units per gram, at least about 1×10⁹ colony-forming units per gram, or at least about 5×10⁹ colony-forming units per gram to at least about 6×10¹⁰ colony-forming units of Methylobacterium per gram, at least about 1×10¹¹ colony-forming units of Methylobacterium per gram, at least about 1×10¹² colony-forming units of Methylobacterium per gram, at least about 1×10¹³ colony-forming units of Methylobacterium per gram, or at least about 5×10¹³ colony-forming units of Methylobacterium per gram of the solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per gram, at least about 5×10⁶ colony-forming units per gram, at least about 1×10⁷ colony-forming units per gram, at least about 5×10⁸ colony-forming units per gram, at least about 1×10⁹ colony-forming units per gram, or at least about 5×10⁹ colony-forming units per gram to at least about 6×10¹⁰ colony-forming units of Methylobacterium per gram, at least about 1×10¹¹ colony-forming units of Methylobacterium per gram, at least about 1×10¹² colony-forming units of Methylobacterium per gram, at least about 1×10¹³ colony-forming units of Methylobacterium per gram, or at least about 5×10¹³ colony-forming units of Methylobacterium per gram of particles in the composition containing the particles that comprise a solid substance wherein a mono-culture or co-culture of Methylobacterium sp. is adhered thereto. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per mL, at least about 5×10⁶ colony-forming units per mL, at least about 1×10⁷ colony-forming units per mL, or at least about 5×10⁸ colony-forming units per mL to at least about 6×10¹⁰ colony-forming units of Methylobacterium per mL in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium sp. adhered to a solid substance is provided therein or grown therein. In certain embodiments, an amount of a composition provided herein that is sufficient to provide for improved tomato production can be a composition with a Methylobacterium titer of at least about 1×10⁶ colony-forming units per mL, at least about 5×10⁶ colony-forming units per mL, at least about 1×10⁷ colony-forming units per mL, or at least about 5×10⁸ colony-forming units per mL to at least about 6×10¹⁰ colony-forming units of Methylobacterium per mL of in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium sp. is provided therein or grown therein.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the Applicants to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the instant disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed, while still obtaining like or similar results, without departing from the scope of the invention**.

Example 1. Culturing of PPFM Strains in a Liquid Growth Media Supplemented with a Solid Substance

The liquid growth medium used to culture the PPFM cultures was a base salts medium supplemented with glycerol, peptone, and diatomaceous earth. The base salts medium used was ammonium mineral salts (AMS) medium. AMS medium contains, per liter, 700 milligrams of dibasic potassium phosphate anhydrous, 540 milligrams of monobasic potassium phosphate anhydrous, one gram of magnesium sulfate heptahydrate, 500 milligrams of ammonium chloride anhydrous, and 200 milligrams of calcium chloride dihydrate.

AMS base medium was prepared from three stock solutions, listed below:

Stock solution I: for one liter at 50X concentration dibasic potassium phosphate, anhydrous 35 grams monobasic potassium phosphate, anhydrous 27 grams Stock solution II: for one liter at 50X concentration magnesium sulfate heptahydrate 50 grams ammonium chloride, anhydrous 25 grams Stock solution III: for one liter at 50X concentration calcium chloride dihydrate 10 grams

Stock solutions I, II, and III were autoclaved separately.

To prepare one liter of liquid AMS medium with glycerol, peptone, and diatomaceous earth, the following were added to 920 ml of distilled water:

20 ml of stock solution I

20 ml of stock solution II

20 ml of stock solution III

20 ml of a 50% glycerol stock solution

10 grams of peptone

2 grams of diatomaceous earth

The resulting solution with suspended diatomaceous earth was sterilized by autoclaving.

Two liters of the above AMS medium were placed into a four-liter flask. Two milliliters of liquid culture PPFMs were added to the media to inoculate. The flask was then placed in an incubated shaker set to 240 RPM and 30 degrees Celsius. The cultures were grown for six days and then stored at 4 degrees Celsius for future use.

Example 2. Seed Inoculation of Tomatoes

Commercial Sweet Olive™ tomato seeds were treated with the PPFM strain NLS0037, and then grown over a time period of about 12-14 days. The titer of strain NLS0037 was 2.0×10⁷ CFU/mL. Two liters of the culture were initially grown in liquid AMS-GP media plus diatomaceous earth at 2 grams/liter (see Example 1). A 100 ml of the culture media was spun down in a centrifuge to form a pellet. The supernatant was then drained and room temperature tap water was added to bring the solution back to its initial volume of 100 ml. Seeds were planted in 100 cell Horticube sheets (an artificial growth media) and treated with 1 ml of solution applied directly to the seed by pipette at the time of planting. The growth media and watering practices simulate a hydroponic treatment. Each experimental unit (control and treated) contained 100 tomato seedlings. The wet weight of each seedling was measured, with the means being reported in Table 2.

TABLE 2 Control and PPFM Treated Tomato Seedling Wet Weights Control wet Treated wet Percentage Confidence Strain weight (mg) weight (mg) increase interval NLS0037, test #1 159 225 42 >95% NLS0037, test #2 170 226 33 >95% NLS0037, test #3 156 194 24 >95%

Example 3. Identification of Nucleic Acid Polymorphisms Present in Methylobacterium that Improve Tomato Production

Whole genome sequencing libraries for the Illumina™ high-throughput sequencing platform are generated for Methylobacterium sp. isolates provided in Table 1 using Illumina TRUSEQ™ or NEXTERA™ DNA sample preparation kits (described on the internet sites res.illumina.com/documents/products/datasheets/datasheet_truseq_dna_sample_prep_kits.pdf and res.illumina.com/documents/products/datasheets/datasheet_nextera_dna_sample_prep.pdf) using the methods described by the manufacturer. The resultant libraries are then subjected to pyrosequencing (Siqueira J F et al. J Oral Microbiol. 2012; 4: 10.3402/jom.v4i0.10743). Raw pyrosequencing-generated genomic sequence data are subjected to adaptor- and quality-based trimming for quality control. Whole-genome Shotgun Sequence Assembly (1) is achieved by assembling quality-passed data using the de novo assembler Velvet (2). For gene finding and annotation, reference training data is leveraged from TIGRFAM (9), Pfam, COG (10), and UniRef100 (11). The rRNAs are identified with RNAmmer (5), protein-coding genes are identified with Glimmer (3) or Maker (6), and tRNAs are identified with tRNAscan-SE (4). Gene functions are assigned with blastx (7), blastp (7), HMMER (8), and InterProScan against comprehensive protein databases described above (Reference Data).

Detection of polymorphisms (SNP or other DNA variations occurring as a result of insertions, deletions, and substitutions (Indels)) in the Methylobacterium sp. isolates of Table 1 is performed with BWA (12) and the Samtools suite (on the internet at samtools.sourceforge.net/), structural variation is identified with BreakDancer (on the internet at breakdancer.sourceforge.net/) and CoGE (on the internet at genomevolution.org/CoGe/). Polymorphisms diagnostic for Methylobacterium that secrete anti-fungal agents are identified by comparisons of the sequences of exemplary Methylobacterium isolate NLS0037 that improve tomato seedling growth but that are absent from one or more Methylobacterium isolates that do not improve tomato. Polymorphisms present in exemplary Methylobacterium isolate NLS0037 that improve tomato production but that are absent in exemplary Methylobacterium isolates that do not improve tomato production are then used to identify other Methylobacterium isolates that improve tomato production.

REFERENCES FOR EXAMPLE 4

-   1. Miller J R, Koren S, Sutton G (2010) Assembly algorithms for     next-generation sequencing data. Genomics 95: 315-327. -   2. Zerbino D R, Birney E (2008) Velvet: algorithms for de novo short     read assembly using de Bruijn graphs. Genome Res 18: 821-829. -   3. Delcher A L, Bratke K A, Powers E C, Salzberg S L (2007)     Identifying bacterial genes and endosymbiont DNA with Glimmer.     Bioinformatics 23: 673-679. -   4. Lowe T M, Eddy S R (1997) tRNAscan-S E: a program for improved     detection of transfer RNA genes in genomic sequence. Nucleic Acids     Res 25: 955-964. -   5. Lagesen K, Hallin P, Rodland E A, Staerfeldt H H, Rognes T, et     al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA     genes. Nucleic Acids Res 35: 3100-3108. -   6. Cantarel B, Korf I, Robb S, et al. (2008) MAKER: An easy-to-use     annotation pipeline designed for emerging model organism genomes.     Genome Research 18: 188-196. -   7. Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, et     al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein     database search programs. Nucleic Acids Res 25: 3389-3402. -   8. Eddy S R (2009) A new generation of homology search tools based     on probabilistic inference. Genome Inform 23: 205-211. -   9. Haft D H, Selengut J D, White 0 (2003) The TIGRFAMs database of     protein families. Nucleic Acids Res 31: 371-373. -   10. Tatusov R L, Fedorova N D, Jackson J D, Jacobs A R, Kiryutin B,     et al. (2003) The COG database: an updated version includes     eukaryotes. BMC Bioinformatics 4: 41. -   11. Suzek B E, Huang H, McGarvey P, Mazumder R, Wu C H (2007)     UniRef: comprehensive and non-redundant UniProt reference clusters.     Bioinformatics 23: 1282-1288. -   12. Li H. and Durbin R. (2009) Fast and accurate short read     alignment with Burrows-Wheeler Transform. Bioinformatics,     25:1754-60.

Example 5. Testing of Additional Methylobacterium Isolates for Stimulation of Tomato Seedling Growth

Methylobacterium isolates NLS0017, NLS0037, NLS0038, and NLS0066 were tested for stimulation of tomato seedling growth essentially as described in Example 2 with the exceptions that dry rather than wet weight of the seedlings was determined and that the amount applied to each seed was 0.25 mL rather than 1 mL. The results of such analyses are shown in Table 3.

TABLE 3 Percent Increase in Seedling Dry Weight relative to control for NLS0017, NLS0037, NLS0038, and NLS0066 treatments Percent Increase in Seedling Dry Weight relative to NLS Isolate control Confidence Interval NLS0017, 16.4% >95%, NLS0037 7.4%  93%, NLS0038 5.6% No statistically significant difference from control NLS0066 23.5% >95%,

The NLS0017 and NLS0066 strains were identified as isolates capable of providing improved tomato seedling growth while NLS0038 was identified as an isolate that did not improve tomato seedling growth in these experiments.

Example 6. Identification of Orthologous Genes Present in Methylobacterium sp. that can Improve Tomato Production

The PPFM strains listed in Table 1 were grown on solid agar media comprising Ammonium Mineral Salts (AMS) plus glycerol and peptone at 30° C. for 5 days, essentially as described in co-assigned U.S. Patent Application Publication No. US20130324407 and incorporated herein by reference in its entirety. Genomic DNA was extracted using MO-BIO (Carlsbad, Calif.) Ultra Clean Microbial DNA Isolation kit, and 1 μg of high quality DNA was used for Illumina Nextera XT library preparation followed by Illumina 2×100 paired-end sequencing on a HiSeq2000 system. Raw Illumina genomic sequence data were subjected to adaptor- and quality-based trimming for quality control. Whole-genome Shotgun Sequence Assembly was achieved by assembling quality-passed data using the de novo assembler SPADES (33). For gene finding and annotation, reference training data was leveraged from TIGRFAM (9), Pfam, COG (10), and UniRef100 (11). The rRNAs were identified with RNAmmer (5), protein-coding genes were identified with Glimmer (3) and Maker (6), and tRNAs were identified with tRNAscan-SE (4). Gene functions were assigned with blastx (7), blastp (7), HMMER (8), and InterProScan against comprehensive protein databases described above (Reference Data). Detection of polymorphisms (SNP or other DNA variations occurring as a result of insertions, deletions, and substitutions (Indels)) in the Methylobacterium sp. isolates was performed with BWA (12) and the Samtools suite (on the internet at samtools.sourceforge.net/) and the Genome Analysis Toolkit (GATK, on the world wide web internet site “broadinstitute.org/gatk/”), structural variation was identified with BreakDancer (on the internet at breakdancer.sourceforge.net/) and CoGE (on the internet at genomevolution.org/CoGe/).

Genes that encoded open reading frames were predicted from the assembled whole genomic sequences of NLS0017, NLS0038, and NLS066 essentially as described above. Within and between genome orthologous genes were clustered using OrthoMCL (available on the world wide web internet site “orthomcl.org/orthomcl/”). Putative functional annotations were assigned to gene products using BLASTP (available on the internet site “blast.ncbi.nlm.nih.gov/Blast.cgi”) against the UniProt database (available on the world wide web internet site “uniprot.org/”). Genes present in individual genomes of NLS0017 and NLS0066 that could improve tomato production (as shown in Example 5) but absent in the genome of NLS0038 that did not improve tomato production (as shown in Example 5) were identified in OrthoMCL clusters using custom software. The encoded proteins found in the Methylobacterium NLS0017 and NLS0066 that could improve tomato production are provided in the sequencing listing as SEQ ID NO: 1-4594. The nucleic acid sequences that encode the proteins of SEQ ID NO: 1-4594 are SEQ ID NO: 4595-9188, respectively. The proteins encoded by genes present in NLS0017 but absent from NLS0038 are provided as SEQ ID NO: 1-2684. The proteins encoded by genes present in NLS0066 but absent from NLS0038 are provided as SEQ ID NO: 2685-4594. Orthologous gene groups representing genes encoding proteins found in the genomes of at least two individual genomes of NLS0017 and NLS0066 that could improve tomato production (as shown in Example 5) but that are absent in the genome of NLS0038 that did not improve tomato production are provided in Table 4. In Table 4, groups of orthologous genes are provided in each row, where the longest sequence and associated unique Seq ID Number are designated as a reference sequence to represent the ortholog cluster (Column 3 of Table 4). The ortholog group identification number is provided in column 1 of Table 4, the closest gene identity based on database comparisons is provided in column 2 of Table 4, and the reference sequence for each ortholog cluster is provided in column 3 of Table 4. Examples of ortholog sequences found in NLS0017 and NLS0066 are provided as SEQ ID NO in Table 4, columns 4, and 5, respectively.

TABLE 4 Orthologous Genes found in NLS0017 and NLS0066 that are absent in NLS0038 Reference. NLS0017 NLS0066 Unique Ortholog Ortholog Ortholog Ortholog Group SEQ ID SEQ ID SEQ ID Identifier Annotation NO: NO: NO: 1107v20141116 membrane protein 1 1 2685 1112v20141116 TRAP-type transport system periplasmic 2 2 2686 component-like protein 1134v20141116 transposase 5 5 2687 1330v20141116 MFS transporter 2688 12 2688 1345v20141116 Hypothetical protein 14 14 2689 1770v20141116 Crp/FNR family transcriptional regulator 18 18 2690 3540v20141116 hypothetical protein 21 21 2691 3605v20141116 porin 22 22 2692 3664v20141116 AraC family transcriptional regulator 24 24 2694 3782v20141116 hypothetical protein 25 25 2695 3796v20141116 Hypothetical protein 2698 26 2698 3837v20141116 hypothetical protein 2700 28 2700 3924v20141116 L-lactate dehydrogenase (cytochrome) 30 30 2702 3930v20141116 hypothetical protein 2703 34 2703 3960v20141116 sulfite:cytochrome C oxidoreductase subunit 2704 36 2704 A 3964v20141116 transposase 2705 37 2705 3974v20141116 putative sulfite:cytochrome c oxidoreductase 38 38 2706 subunit B 4022v20141116 hypothetical protein 42 42 2707 4025v20141116 hypothetical protein 43 43 2708 MexAM1_META1p1708 4057v20141116 major facilitator superfamily protein 49 49 2709 4058v20141116 hypothetical protein 50 50 2710 4061v20141116 hypothetical protein 51 51 2711 4068v20141116 pyruvate kinase 52 52 2712 4075v20141116 hypothetical protein 2713 53 2713 4082v20141116 FAD-dependent oxidoreductase 55 55 2714 4084v20141116 hypothetical protein 2715 57 2715 4106v20141116 ECF subfamily RNA polymerase sigma-24 2716 58 2716 factor 4113v20141116 short-chain dehydrogenase/reductase SDR 59 59 2717 4124v20141116 MarR family transcriptional regulator 2718 60 2718 4146v20141116 two component transcriptional regulator 2721 61 2721 4147v20141116 hypothetical protein 62 62 2722 4155v20141116 hypothetical protein Mrad2831_1363 64 64 2723 4162v20141116 cobalt-containing nitrile hydratase subunit 65 65 2726 alpha 4163v20141116 regulatory protein 67 67 2727 4165v20141116 formyl transferase 2728 69 2728 4192v20141116 aldo/keto reductase 72 72 2729 4214v20141116 aliphatic nitrilase 74 74 2730 4228v20141116 hypothetical protein 75 75 2731 4269v20141116 TonB-dependent siderophore receptor 2732 77 2732 4288v20141116 ABC transporter-like protein 84 84 2734 4325v20141116 FAD-dependent oxidoreductase 91 91 2736 4327v20141116 hypothetical protein 92 92 2737 4335v20141116 hypothetical protein Mrad2831_6489 2738 93 2738 4353v20141116 transposase partial 95 95 2739 4354v20141116 magnesium transporter 96 96 2740 4356v20141116 spermidine/putrescine ABC transporter 2741 97 2741 ATP-binding protein 4376v20141116 hypothetical protein 2743 103 2743 4407v20141116 hypothetical protein 107 107 2744 4409v20141116 Asp/Glu racemase 2745 109 2745 4410v20141116 binding-protein-dependent transport system 110 110 2746 inner membrane protein 4412v20141116 MFS transporter 2748 111 2748 4421v20141116 hypothetical protein 112 112 2751 4424v20141116 hypothetical protein 2752 113 2752 4442v20141116 sulfonate ABC transporter ATP-binding 117 117 2753 lipoprotein 4460v20141116 partition protein 121 121 2755 4464v20141116 extracellular ligand-binding receptor 2756 123 2756 4466v20141116 hypothetical protein 2757 124 2757 4482v20141116 hypothetical protein 2758 126 2758 4499v20141116 sulfolactate dehydrogenase 127 127 2760 4505v20141116 hypothetical protein 2761 130 2761 4506v20141116 ABC transporter-like protein 131 131 2762 4507v20141116 hypothetical protein 2763 132 2763 4508v20141116 inner-membrane translocator 133 133 2764 4509v20141116 branched-chain amino acid transporter 134 134 2765 permease subunit LivH 4518v20141116 hypothetical protein 135 135 2766 4519v20141116 Hypothetical protein 2767 136 2767 4520v20141116 MFS transporter 137 137 2770 4522v20141116 D-amino acid dehydrogenase small subunit 2771 138 2771 4525v20141116 allantoate amidohydrolase 141 141 2772 4534v20141116 ABC transporter ATP-binding protein 143 143 2775 4537v20141116 beta-lactamase 2776 144 2776 4542v20141116 4-phosphopantetheinyl transferase 2777 146 2777 4546v20141116 hypothetical protein 147 147 2778 4562v20141116 hypothetical protein 2779 151 2779 4563v20141116 urea ABC transporter permease 2780 153 2780 4564v20141116 amino acid ABC transporter permease 154 154 2782 4566v20141116 branched-chain amino acid ABC transporter 155 155 2784 substrate-binding protein 4574v20141116 hypothetical protein 157 157 2787 4579v20141116 ABC transporter substrate-binding protein 2788 160 2788 4582v20141116 UDP-3-0-acyl N-acetylglucosamine 2789 162 2789 deacetylase 4584v20141116 MFS transporter 164 164 2790 4586v20141116 hypothetical protein 165 165 2791 4588v20141116 30S ribosomal protein S13 2792 166 2792 4601v20141116 nitrate ABC transporter substrate-binding 2794 167 2794 protein 4607v20141116 glutaminase 2795 170 2795 4610v20141116 hypothetical protein 171 171 2796 4612v20141116 glyoxalase/bleomycin resistance 2797 172 2797 protein/dioxygenase 4613v20141116 shikimate kinase 173 173 2798 4614v20141116 hypothetical protein 174 174 2799 4615v20141116 putative sulfite oxidase subunit YedY 175 175 2800 4616v20141116 RNA polymerase sigma factor 176 176 2801 4619v20141116 hypothetical protein Mrad2831_0815 177 177 2802 4624v20141116 hypothetical protein Mnod_0273 179 179 2803 4627v20141116 ferric reductase 180 180 2805 4628v20141116 hypothetical protein 181 181 2806 4634v20141116 hypothetical protein Mrad2831_4175 2807 185 2807 4642v20141116 hypothetical protein 187 187 2808 4644v20141116 hypothetical protein 2809 188 2809 4646v20141116 hypothetical protein 189 189 2810 4648v20141116 hypothetical protein 2811 190 2811 4652v20141116 response regulator receiver protein 192 192 2812 4654v20141116 hypothetical protein 193 193 2813 MexAM1_META1p3794 4656v20141116 HupE/UreJ protein 195 195 2814 4657v20141116 hypothetical protein 2815 196 2815 4659v20141116 hypothetical protein 198 198 2816 4661v20141116 cupin 2817 200 2817 4663v20141116 hypothetical protein 2818 201 2818 4665v20141116 hypothetical protein 2819 202 2819 4676v20141116 response regulator receiver sensor hybrid 204 204 2820 histidine kinase 4681v20141116 hypothetical protein 205 205 2821 4683v20141116 hypothetical protein 2822 206 2822 4684v20141116 hypothetical protein M446_2722 2823 207 2823 4686v20141116 hypothetical protein 208 208 2824 4687v20141116 peptidase S14 ClpP 209 209 2825 4688v20141116 hypothetical protein 210 210 2826 4689v20141116 hypothetical protein 2827 211 2827 4690v20141116 LysR family transcriptional regulator 212 212 2828 4691v20141116 hypothetical protein 213 213 2829 4692v20141116 hypothetical protein 2830 214 2830 4694v20141116 hypothetical protein 2831 215 2831 4695v20141116 hypothetical protein M446_0699 217 217 2832 4696v20141116 MazF family transcriptional regulator 218 218 2833 4697v20141116 hypothetical protein Mnod_6017 219 219 2834 4699v20141116 Fmn-binding pyridoxamine 5-phosphate 2835 220 2835 oxidase 4704v20141116 siderophore biosynthesis protein 2836 222 2836 4705v20141116 hypothetical protein 2837 223 2837 4706v20141116 sorbosone dehydrogenase 2838 224 2838 4710v20141116 sensor histidine kinase 225 225 2839 4715v20141116 peptidase 2840 226 2840 4716v20141116 metallophosphoesterase 227 227 2841 4720v20141116 nitrile hydratase subunit beta 2842 228 2842 4721v20141116 hypothetical protein 229 229 2843 4723v20141116 hypothetical protein 230 230 2844 4725v20141116 NAD-dependent epimerase/dehydratase 2845 231 2845 4743v20141116 AsnC family transcriptional regulator 233 233 2848 4750v20141116 hypothetical protein 2849 234 2849 4751v20141116 hypothetical protein 2850 235 2850 4752v20141116 hypothetical protein 2851 236 2851 4756v20141116 hypothetical protein 2852 237 2852 4757v20141116 hypothetical protein 2853 238 2853 4759v20141116 peptidase M20 2854 239 2854 4766v20141116 iron reductase 2855 242 2855 4767v20141116 hypothetical protein 243 243 2856 4771v20141116 AsnC family transcriptional regulator 244 244 2857 4772v20141116 transcriptional regulator 245 245 2858 4774v20141116 hypothetical protein 2859 246 2859 4789v20141116 fusaric acid resistance protein 247 247 2861 4796v20141116 pyruvate dehydrogenase 249 249 2862 4800v20141116 GntR family transcriptional regulator 2863 250 2863 4801v20141116 hypothetical protein 251 251 2864 4802v20141116 hypothetical protein 252 252 2865 4806v20141116 Protein of unknown function DUF2474 2866 255 2866 4811v20141116 2 4-dihydroxyhept-2-ene-1 7-dioic acid 258 258 2868 aldolase 4814v20141116 hypothetical protein 259 259 2869 4834v20141116 DltE 263 263 2873 4838v20141116 methyl-accepting chemotaxis 264 264 2875 receptor/sensory transducer 4842v20141116 hypothetical protein 2876 266 2876 4843v20141116 ABC transporter substrate-binding protein 267 267 2877 4844v20141116 ABC transporter permease 268 268 2878 4847v20141116 hypothetical protein 270 270 2879 4849v20141116 two component LuxR family transcriptional 272 272 2880 regulator 4850v20141116 Peptidase family M20/M25/M40 protein 273 273 2881 4851v20141116 peptide ABC transporter permease 2882 274 2882 4877v20141116 DoxX family protein 2886 278 2886 4883v20141116 binding-protein-dependent transport system 280 280 2887 inner membrane protein 4884v20141116 methionine ABC transporter ATP-binding 281 281 2888 protein 4885v20141116 hypothetical protein 282 282 2889 4907v20141116 Glucose-methanol-choline (GMC) 286 286 2892 oxidoreductase:NAD binding site 4910v20141116 LysR family transcriptional regulator 289 289 2893 4911v20141116 orotate phosphoribosyltransferase 291 291 2894 4912v20141116 hypothetical protein 2895 292 2895 4917v20141116 membrane protein 295 295 2896 4918v20141116 RND family efflux transporter MFP subunit 2897 296 2897 4920v20141116 hypothetical protein 2899 298 2899 4921v20141116 hypothetical protein 299 299 2900 4923v20141116 NLPA lipoprotein 301 301 2901 4947v20141116 hypothetical protein 303 303 2906 4954v20141116 LuxR family transcriptional regulator 2907 308 2907 4958v20141116 cupin 2908 311 2908 4961v20141116 amino acid ABC transporter 312 312 2910 4963v20141116 response regulator receiver protein 314 314 2911 4983v20141116 Hypothetical protein 2914 316 2914 4986v20141116 hypothetical protein 317 317 2916 4989v20141116 peptidase S9 319 319 2917 4992v20141116 N-acetyltransferase GCN5 320 320 2918 4993v20141116 glutamate carboxypeptidase 2919 321 2919 4995v20141116 hypothetical protein Mchl_4780 322 322 2920 5001v20141116 nitrate reductase 2921 325 2921 5016v20141116 hypothetical protein 327 327 2923 5017v20141116 diguanylate cyclase 2924 328 2924 5018v20141116 hypothetical protein 329 329 2925 5019v20141116 hypothetical protein 330 330 2926 5028v20141116 hypothetical protein 2928 335 2928 5030v20141116 ABC transporter permease 337 337 2929 5034v20141116 carbohydrate-selective porin OprB 339 339 2930 5036v20141116 hypothetical protein 340 340 2931 5039v20141116 hypothetical protein 342 342 2932 5070v20141116 amidase 347 347 2939 5071v20141116 type I protein secretion ATP-binding protein 348 348 2940 HlyB 5073v20141116 hypothetical protein 349 349 2941 5075v20141116 gamma carboxymuconolactone 352 352 2942 decarboxylase 5076v20141116 D-serine dehydratase 2943 353 2943 5085v20141116 hypothetical protein Mchl_4781 2944 359 2944 5092v20141116 ABC transporter substrate-binding protein 365 365 2945 5099v20141116 MarR family transcriptional regulator 368 368 2947 5121v20141116 histidine kinase 371 371 2949 5124v20141116 DSBA oxidoreductase 373 373 2950 5125v20141116 methyl-accepting chemotaxis sensory 2951 374 2951 transducer 5129v20141116 crotonase 376 376 2952 5133v20141116 amino acid ABC transporter substrate- 379 379 2953 binding protein 5137v20141116 ferredoxin subunit of nitrite reductase and 380 380 2954 ring-hydroxylating dioxygenase 5138v20141116 ABC transporter 2955 381 2955 5139v20141116 peptide ABC transporter 382 382 2956 5182v20141116 hypothetical protein 2962 386 2962 5190v20141116 chromosome partitioning protein ParA 391 391 2965 5196v20141116 secretion protein HlyD family protein 397 397 2966 5197v20141116 hypothetical protein 398 398 2967 5199v20141116 XRE family transcriptional regulator 2969 399 2969 5203v20141116 COG0346: Lactoylglutathione lyase and 2970 402 2970 related lyases 5204v20141116 COG3386: Gluconolactonase partial 403 403 2971 5207v20141116 ABC transporter permease 2972 405 2972 5208v20141116 ABC transporter permease 406 406 2973 5209v20141116 dihydroorotase 2974 407 2974 5236v20141116 epoxide hydrolase 2977 408 2977 5238v20141116 OmpA/MotB domain-containing protein 2978 410 2978 5242v20141116 hypothetical protein 411 411 2979 5243v20141116 hypothetical protein 412 412 2980 5244v20141116 endoribonuclease L-PSP 413 413 2982 5245v20141116 molybdenum cofactor biosysynthesis protein 414 414 2983 5255v20141116 peptide ABC transporter permease 416 416 2984 5256v20141116 sugar ABC transporter substrate-binding 417 417 2985 protein 5257v20141116 hypothetical protein 2986 418 2986 5333v20141116 xanthine dehydrogenase 2991 421 2991 5352v20141116 hypothetical protein 430 430 2993 5357v20141116 ferredoxin 433 433 2994 5365v20141116 3-isopropylmalate dehydrogenase 439 439 2995 5371v20141116 methyl-accepting chemotaxis sensory 442 442 2996 transducer 5372v20141116 group 1 glycosyl transferase 2997 444 2997 5373v20141116 chemotaxis protein CheW 2998 445 2998 5422v20141116 alanine racemase domain-containing protein 451 451 3009 5423v20141116 ArsR family transcriptional regulator 452 452 3010 5426v20141116 hypothetical protein 453 453 3011 5428v20141116 hypothetical protein 455 455 3012 5430v20141116 HxlR family transcriptional regulator 3013 457 3013 5433v20141116 peptidase C14 3014 460 3014 5434v20141116 hypothetical protein 461 461 3015 5436v20141116 LysR family transcriptional regulator 3016 463 3016 5442v20141116 hypothetical protein 3017 466 3017 5443v20141116 hypothetical protein 467 467 3018 5444v20141116 hypothetical protein Mext_0240 468 468 3019 5445v20141116 type 11 methyltransferase 469 469 3020 5446v20141116 phosphoglycerate mutase 470 470 3021 5447v20141116 myo-inositol-1-phosphate synthase 3022 471 3022 5448v20141116 chemotaxis protein CheA 3023 472 3023 5450v20141116 NAD-dependent epimerase/dehydratase 3024 474 3024 5451v20141116 radical SAM protein 475 475 3025 5452v20141116 Hypothetical protein 3026 476 3026 5453v20141116 hypothetical protein Mrad2831_1317 3027 477 3027 5454v20141116 response regulator receiver modulated CheB 3028 478 3028 methylesterase 5500v20141116 porin 484 484 3038 5506v20141116 hypothetical protein 3040 486 3040 5507v20141116 hypothetical protein 487 487 3041 5508v20141116 hypothetical protein Mpop_0725 3042 488 3042 5509v20141116 hypothetical protein 3043 489 3043 5510v20141116 hypothetical protein 3044 490 3044 5516v20141116 hypothetical protein Mpop_1265 491 491 3046 5517v20141116 chromosome partitioning protein 492 492 3047 5569v20141116 metal dependent phosphohydrolase 495 495 3053 5573v20141116 hypothetical protein Mext_1867 497 497 3054 5580v20141116 hypothetical protein Mpop_2258 3056 500 3056 5583v20141116 hypothetical protein Mpop_3020 3057 502 3057 5585v20141116 hypothetical protein Mpop_0722 503 503 3058 5586v20141116 hypothetical protein Mpop_0723 504 504 3059 5589v20141116 XRE family transcriptional regulator 505 505 3060 5598v20141116 PBS lyase 3061 510 3061 5599v20141116 chemotaxis protein CheY 511 511 3062 5647v20141116 GDP-mannose 4 6-dehydratase 3071 516 3071 5658v20141116 hypothetical protein Mrad2831_3432 517 517 3072 5662v20141116 hypothetical protein 520 520 3074 5665v20141116 Hypothetical protein 3075 522 3075 5668v20141116 cytochrome B561 523 523 3076 5670v20141116 Phosphoribosylaminoimidazole- 3077 525 3077 succinocarboxamide synthase 5673v20141116 chemotaxis sensory transducer protein 527 527 3078 5778v20141116 hypothetical protein 3089 548 3089 5784v20141116 hypothetical protein 3090 552 3090 5785v20141116 hypothetical protein 3091 554 3091 5786v20141116 Sulfur oxidation protein SoxZ 557 557 3092 5787v20141116 sulfur oxidation cytochrome c protein SoxA 558 558 3093 5788v20141116 MFS transporter 560 560 3094 5789v20141116 mandelate racemase/muconate lactonizing 3095 561 3095 protein 5792v20141116 PAS domain-containing protein 563 563 3096 5793v20141116 sugar transporter 3097 564 3097 5843v20141116 Hypothetical protein 569 569 3106 5849v20141116 hypothetical protein Mrad2831_5253 3107 575 3107 5851v20141116 chemotaxis protein 576 576 3108 5852v20141116 AsnC family transcriptional regulator 3109 577 3109 5854v20141116 hypothetical protein 3110 578 3110 5855v20141116 hypothetical protein 3111 579 3111 5856v20141116 NAD-glutamate dehydrogenase 3112 580 3112 5857v20141116 hypothetical protein 581 581 3113 5860v20141116 transcriptional regulator XRE family 3114 584 3114 5862v20141116 2-nitropropane dioxygenase 3116 585 3116 5926v20141116 dioxygenase 3126 588 3126 5929v20141116 gamma-glutamyltransferase 589 589 3128 5930v20141116 RND efflux system outer membrane 3129 590 3129 lipoprotein NodT family 5936v20141116 Hypothetical protein 592 592 3130 5938v20141116 Cytochrome c class I 593 593 3131 5939v20141116 hypothetical protein 3132 594 3132 5988v20141116 extracellular ligand-binding receptor 600 600 3144 5993v20141116 hypothetical protein Mrad2831_6386 604 604 3145 6001v20141116 transporter 606 606 3147 6006v20141116 Leu/Ile/Val-binding family protein 3148 608 3148 6007v20141116 hypothetical protein 609 609 3149 6010v20141116 hypothetical protein Mrad2831_1535 610 610 3150 6012v20141116 hypothetical protein 3151 613 3151 6014v20141116 hypothetical protein 614 614 3152 6016v20141116 family 5 extracellular solute-binding protein 616 616 3153 6017v20141116 acyl-CoA dehydrogenase 3154 617 3154 6021v20141116 diguanylate cyclase 618 618 3157 6023v20141116 hydroxymethylglutaryl-CoA lyase 3158 619 3158 6024v20141116 hypothetical protein 3159 620 3159 6026v20141116 NAD-binding 3-hydroxyacyl-CoA 3160 621 3160 dehydrogenase 6027v20141116 L-carnitine dehydratase/bile acid-inducible 622 622 3161 protein F 6093v20141116 Fe—S type tartrate/fumarate subfamily 625 625 3166 hydro-lyase subunit alpha 6095v20141116 hypothetical protein 3167 626 3167 6101v20141116 glutathione S-transferase 627 627 3168 6115v20141116 NAD-dependent epimerase/dehydratase 629 629 3171 6116v20141116 sorbosone dehydrogenase 630 630 3172 6117v20141116 cytochrome C 3173 631 3173 6118v20141116 hypothetical protein Mrad2831_0725 632 632 3174 6119v20141116 serine/threonine protein phosphatase 3175 633 3175 6124v20141116 hypothetical protein 636 636 3176 6125v20141116 malate synthase G 3177 637 3177 6126v20141116 LysR family transcriptional regulator 3178 638 3178 6130v20141116 alanine racemase 641 641 3179 6131v20141116 3-hydroxyisobutyrate dehydrogenase 3180 642 3180 6133v20141116 acyl carrier protein 644 644 3181 6134v20141116 hypothetical protein 645 645 3182 6135v20141116 hypothetical protein 3183 646 3183 6137v20141116 hypothetical protein 3184 648 3184 6142v20141116 L-carnitine dehydratase/bile acid-inducible 649 649 3185 protein F 6143v20141116 acetolactate synthase 3186 650 3186 6188v20141116 GntR family transcriptional regulator 656 656 3194 6193v20141116 hypothetical protein 657 657 3195 6194v20141116 FAD linked oxidase domain-containing 658 658 3196 protein 6200v20141116 TRAP transporter solute receptor TAXI 3197 662 3197 family protein 6201v20141116 hypothetical protein Mext_2439 3198 663 3198 6202v20141116 alpha/beta hydrolase 664 664 3199 6203v20141116 electron transporter 3200 665 3200 6204v20141116 hypothetical protein 666 666 3201 6205v20141116 hypothetical protein 667 667 3202 6206v20141116 amine oxidase 3203 668 3203 6207v20141116 2-hydroxyacid dehydrogenase 669 669 3204 6209v20141116 hypothetical protein 3205 670 3205 6210v20141116 Bcr/CflA subfamily drug resistance 3206 671 3206 transporter 6214v20141116 acyl-CoA dehydrogenase domain-containing 3207 672 3207 protein 6219v20141116 acyl-CoA dehydrogenase 674 674 3208 6220v20141116 succinate-semialdehyde dehydrogenase 675 675 3209 6221v20141116 dihydrodipicolinate synthetase 676 676 3210 6225v20141116 hypothetical protein 680 680 3211 6226v20141116 potassium-transporting ATPase subunit B 3212 681 3212 6229v20141116 type III effector Hrp-dependent protein 3213 682 3213 6230v20141116 LacI family transcriptional regulator 3214 683 3214 6231v20141116 putative aldolase 684 684 3215 6233v20141116 glycosyl transferase family 1 685 685 3216 6235v20141116 hypothetical protein 687 687 3217 6236v20141116 serine/threonine dehydratase 688 688 3218 6238v20141116 hypothetical protein 689 689 3219 6239v20141116 oxidase 690 690 3220 6241v20141116 SPW repeat-containing protein 3221 693 3221 6243v20141116 tartronate semialdehyde reductase 694 694 3222 6245v20141116 ABC transporter permease 695 695 3223 6246v20141116 binding-protein-dependent transport system 3224 696 3224 inner membrane protein 6247v20141116 ABC transporter substrate-binding protein 3225 697 3225 6248v20141116 spermidine/putrescine ABC transporter 698 698 3226 ATPase 6249v20141116 dihydropyrimidinase 699 699 3227 6250v20141116 poly-beta-hydroxybutyrate polymerase 700 700 3228 6253v20141116 aldo/keto reductase 3229 702 3229 6254v20141116 circadian phase modifier CpmA 703 703 3230 6325v20141116 hypothetical protein 709 709 3231 6328v20141116 GCN5 family acetyltransferase 712 712 3232 6329v20141116 MFS transporter 713 713 3233 6331v20141116 major facilitator superfamily protein 715 715 3234 6332v20141116 L-carnitine dehydratase/bile acid-inducible 3235 716 3235 protein F 6333v20141116 hypothetical protein 3236 717 3236 6334v20141116 dihydroxy-acid dehydratase 3237 718 3237 6337v20141116 3-hydroxyisobutyrate dehydrogenase 3238 721 3238 6340v20141116 2-dehydropantoate 2-reductase 724 724 3239 6343v20141116 cytochrome C 726 726 3240 6346v20141116 hypothetical protein 3241 729 3241 6347v20141116 alanine racemase 730 730 3242 6348v20141116 hypothetical protein 3243 731 3243 6351v20141116 D-galactarate dehydratase 733 733 3244 6353v20141116 LysR family transcriptional regulator 734 734 3245 6358v20141116 3-hydroxy-2-methylbutyryl-CoA 3246 735 3246 dehydrogenase 6413v20141116 flagellar protein FlgA 736 736 3254 6414v20141116 altronate dehydratase 737 737 3255 6415v20141116 D-isomer specific 2-hydroxyacid 738 738 3256 dehydrogenase NAD-binding subunit 6423v20141116 flp fap pilin component 739 739 3257 6430v20141116 inner-membrane translocator 744 744 3258 6431v20141116 sn-glycerol-3-phosphate ABC transporter 745 745 3259 substrate-binding protein 6432v20141116 hypothetical protein 746 746 3260 6435v20141116 family 5 extracellular solute-binding protein 3261 748 3261 6438v20141116 hypothetical protein 3262 749 3262 6440v20141116 gamma-glutamyltransferase 751 751 3263 6441v20141116 prolyl-tRNA synthetase 752 752 3264 6444v20141116 HAD-superfamily phosphatase subfamily 3265 753 3265 IIIC domain protein 6445v20141116 4-methylmuconolactone transporter 3266 754 3266 6446v20141116 GCN5 family acetyltransferase 755 755 3267 6449v20141116 hypothetical protein 757 757 3268 6452v20141116 diguanylate cyclase/phosphodiesterase 759 759 3269 6453v20141116 putative alkaline phosphatase 760 760 3270 6454v20141116 binding-protein-dependent transport system 761 761 3271 inner membrane protein 6456v20141116 hypothetical protein 3272 763 3272 6457v20141116 amidase 3273 764 3273 6460v20141116 iron-containing alcohol dehydrogenase 3274 765 3274 6461v20141116 acetyl-CoA acetyltransferase 766 766 3275 6462v20141116 pimeloyl-CoA dehydrogenase large subunit 767 767 3276 6463v20141116 acyl-CoA dehydrogenase 768 768 3277 6465v20141116 IclR family transcriptional regulator 769 769 3278 6466v20141116 hypothetical protein Mnod_2193 770 770 3279 6469v20141116 acetylornithine deacetylase 3280 772 3280 6578v20141116 hypothetical protein 775 775 3289 6580v20141116 ABC transporter substrate-binding protein 776 776 3290 6581v20141116 hypothetical protein 777 777 3291 6586v20141116 dimethylmenaquinone methyltransferase 3292 779 3292 6589v20141116 hypothetical protein 3293 781 3293 6594v20141116 GntR family transcriptional regulator 3295 785 3295 6595v20141116 LysR family transcriptional regulator 786 786 3296 6600v20141116 methylase 789 789 3297 6605v20141116 4-phytase 792 792 3298 6609v20141116 amino acid ABC transporter substrate- 3299 796 3299 binding protein 6610v20141116 ABC transporter permease 797 797 3300 6611v20141116 hypothetical protein 798 798 3301 6673v20141116 peptide ABC transporter substrate-binding 800 800 3316 protein 6674v20141116 ABC transporter ATP-binding protein 3317 801 3317 6679v20141116 MucR family transcriptional regulator 802 802 3318 6681v20141116 XRE family transcriptional regulator 804 804 3319 6682v20141116 hypothetical protein 805 805 3320 6685v20141116 hypothetical protein 808 808 3321 6688v20141116 hypothetical protein 3322 811 3322 6689v20141116 catalase 3323 812 3323 6690v20141116 hypothetical protein 3324 813 3324 6699v20141116 hypothetical protein Mrad2831_3163 3325 822 3325 6700v20141116 hypothetical protein 823 823 3326 6702v20141116 hypothetical protein 825 825 3327 6703v20141116 fatty acid metabolism AMP-binding protein 3328 826 3328 6704v20141116 hypothetical protein 3329 827 3329 6706v20141116 DeoR family transcriptional regulator 829 829 3330 6707v20141116 glucarate dehydratase 3331 830 3331 6708v20141116 PAS/PAC sensor protein 831 831 3332 6709v20141116 hypothetical protein 832 832 3333 6710v20141116 hypothetical protein 3334 833 3334 6711v20141116 hypothetical protein 3335 834 3335 6712v20141116 hypothetical protein Mrad2831_5112 835 835 3336 6714v20141116 alcohol dehydrogenase 836 836 3338 6715v20141116 hypothetical protein 3339 837 3339 6716v20141116 hypothetical protein 838 838 3340 6718v20141116 hypothetical protein 3341 840 3341 6719v20141116 hypothetical protein 841 841 3342 6721v20141116 hypothetical protein Mrad2831_3655 3343 843 3343 6722v20141116 hypothetical protein Mrad2831_0445 844 844 3344 6724v20141116 hypothetical protein 3345 845 3345 6729v20141116 hypothetical protein 850 850 3346 6731v20141116 photo system reaction center subunit H 3347 853 3347 6732v20141116 hypothetical protein Mrad2831_3817 854 854 3348 6736v20141116 hypothetical protein Mrad2831_2399 856 856 3349 6737v20141116 hypothetical protein 3350 857 3350 6738v20141116 hypothetical protein 3351 858 3351 6742v20141116 epimerase 3352 861 3352 6743v20141116 hypothetical protein 3353 862 3353 6800v20141116 NAD-binding D-isomer specific 2- 3363 867 3363 hydroxyacid dehydrogenase 6806v20141116 ArsR family transcriptional regulator 3364 872 3364 6807v20141116 cysteine dioxygenase 3365 873 3365 6808v20141116 hypothetical protein 874 874 3366 6809v20141116 (2Fe—2S)-binding domain-containing protein 875 875 3367 6810v20141116 aldehyde dehydrogenase 876 876 3368 6811v20141116 hypothetical protein Mnod_6032 3369 877 3369 6812v20141116 histone deacetylase 878 878 3370 6818v20141116 hypothetical protein 882 882 3371 6896v20141116 hypothetical protein Mrad2831_5186 3379 894 3379 6903v20141116 diguanylate cyclase 897 897 3380 6907v20141116 translation initiation factor IF-2 3381 899 3381 6909v20141116 hypothetical protein 3382 902 3382 6921v20141116 acetyl-CoA carboxylase 913 913 3383 6927v20141116 binding-protein-dependent transport system 917 917 3384 inner membrane protein 6936v20141116 hypothetical protein 924 924 3385 6938v20141116 hypothetical protein 926 926 3386 6940v20141116 domain of unknown function family protein 3387 928 3387 6943v20141116 transposase IS4 family protein 930 930 3388 7006v20141116 binding-protein-dependent transport system 3396 932 3396 inner membrane protein 7015v20141116 hypothetical protein 940 940 3397 7017v20141116 hypothetical protein 942 942 3398 7023v20141116 type III restriction endonuclease subunit R 3399 947 3399 7027v20141116 LysR family transcriptional regulator 3400 950 3400 7029v20141116 hypothetical protein 951 951 3402 7040v20141116 hypothetical protein 963 963 3403 7042v20141116 arginine ABC transporter ATP-binding 965 965 3404 protein 7048v20141116 glyoxalase 969 969 3405 7050v20141116 urea ABC transporter ATP-binding protein 3406 971 3406 UrtD 7051v20141116 urea ABC transporter ATP-binding protein 972 972 3407 UrtE 7053v20141116 hypothetical protein 973 973 3408 7054v20141116 GntR family transcriptional regulator 3409 974 3409 7127v20141116 hypothetical protein Mnod_6985 3416 983 3416 7152v20141116 hydratase/decarboxylase 3418 1001 3418 7153v20141116 putative membrane protein 1002 1002 3419 7155v20141116 Lipopolysaccharide biosynthesis protein-like 3420 1004 3420 protein 7156v20141116 aldolase 1005 1005 3421 7163v20141116 glycerophosphoryl diester phosphodiesterase 1011 1011 3422 7180v20141116 adenylate cyclase 1027 1027 3423 7189v20141116 hypothetical protein 1034 1034 3425 VOLCADRAFT_119358 7190v20141116 Glyoxalase/Bleomycin resistance 3426 1035 3426 protein/Dioxygenase superfamily 7200v20141116 competence protein ComEA 1046 1046 3427 7201v20141116 serine/threonine dehydratase 1047 1047 3428 7202v20141116 serine--glyoxylate aminotransferase 1048 1048 3429 7211v20141116 cytochrome C oxidase subunit III 1057 1057 3430 7290v20141116 succinate dehydrogenase and fumarate 3435 1064 3435 reductase iron-sulfur protein 7291v20141116 succinate dehydrogenase membrane anchor 1065 1065 3436 7292v20141116 succinate dehydrogenase cytochrome b 1066 1066 3437 subunit 7293v20141116 L(+)-tartrate or fumarate dehydratase 1067 1067 3438 subunit beta 7294v20141116 fumarate reductase 1068 1068 3439 7295v20141116 YCII-like protein 1069 1069 3440 7299v20141116 glycosyltransferase family 2 3441 1071 3441 7310v20141116 ABC transporter substrate-binding protein 3442 1074 3442 7311v20141116 glutathione ABC transporter permease GsiD 1075 1075 3443 7312v20141116 oligopeptide/dipeptide ABC transporter 1076 1076 3444 ATPase 7313v20141116 ABC transporter-like protein 1077 1077 3445 7314v20141116 sodium:calcium antiporter 3446 1078 3446 7315v20141116 methionyl-tRNA formyltransferase 1079 1079 3447 7317v20141116 hypothetical protein 1080 1080 3449 7326v20141116 glycosyl transferase family protein 1088 1088 3450 7331v20141116 hypothetical protein Mrad2831_4126 3452 1090 3452 7332v20141116 4-oxalomesaconate hydratase 1091 1091 3453 7340v20141116 hypothetical protein FAES_2018 1098 1098 3454 7341v20141116 hypothetical protein M446_1279 3455 1099 3455 7343v20141116 hypothetical protein 3456 1101 3456 7349v20141116 diguanylate cyclase 3457 1107 3457 7350v20141116 hypothetical protein 1108 1108 3458 7354v20141116 acetyl-CoA synthetase 1111 1111 3459 7355v20141116 phenylacetic acid degradation protein 1112 1112 3460 7356v20141116 alcohol dehydrogenase 3461 1113 3461 7357v20141116 nitrate/sulfonate/bicarbonate ABC 3462 1114 3462 transporter periplasmic ligand-binding protein 7358v20141116 nitrate ABC transporter permease 3463 1115 3463 7360v20141116 hypothetical protein 1117 1117 3464 7363v20141116 hypothetical protein Mrad2831_1876 1120 1120 3465 7365v20141116 hypothetical protein Mrad2831_6026 3467 1121 3467 7368v20141116 enoyl-CoA hydratase/isomerase 3468 1124 3468 7370v20141116 nitrate ABC transporter ATPase 1126 1126 3469 7372v20141116 hypothetical protein 3470 1128 3470 7472v20141116 hypothetical protein Mext_2440 1131 1131 3482 7478v20141116 porin 3483 1132 3483 7485v20141116 branched-chain amino acid ABC transporter 1138 1138 3484 permease 7538v20141116 RND family efflux transporter MFP subunit 3488 1185 3488 7554v20141116 phosphoheptose isomerase 3490 1198 3490 7555v20141116 GHMP kinase 3491 1199 3491 7570v20141116 IclR family transcriptional regulator 1215 1215 3492 7695v20141116 extracellular ligand-binding receptor 1233 1233 3506 7707v20141116 metal-dependent phosphohydrolase 1243 1243 3508 7711v20141116 2-hydroxyacid dehydrogenase 3509 1247 3509 7729v20141116 amino acid ABC transporter 3510 1264 3510 7730v20141116 GntR family transcriptional regulator 1265 1265 3511 7750v20141116 alpha-amylase 1283 1283 3512 7856v20141116 hypothetical protein 3528 1303 3528 7868v20141116 hypothetical protein 1313 1313 3529 7877v20141116 hypothetical protein Mchl_0532 3530 1319 3530 7879v20141116 glycosyl transferase 3531 1321 3531 7885v20141116 binding-protein-dependent transport system 1327 1327 3532 inner membrane protein 7888v20141116 hypothetical protein Mrad2831_1281 3533 1330 3533 7890v20141116 taurine ABC transporter permease 1332 1332 3534 7913v20141116 D-lactate dehydrogenase 1351 1351 3536 8053v20141116 acetyltransferase 1371 1371 3558 8080v20141116 hypothetical protein 1397 1397 3560 8092v20141116 ABC transporter inner membrane protein 3561 1406 3561 8093v20141116 ABC transporter 1407 1407 3562 8094v20141116 nitrate/sulfonate/bicarbonate ABC 3563 1408 3563 transporter 8109v20141116 Hypothetical protein 1423 1423 3564 8113v20141116 adenylate/guanylate cyclase 3565 1427 3565 8114v20141116 polysaccharide deacetylase 1428 1428 3566 8300v20141116 Holliday junction DNA helicase RuvB 1471 1471 3588 8301v20141116 None 3589 1472 3589 8310v20141116 monooxygenase 1480 1480 3590 8313v20141116 GDP-L-fucose synthase 1483 1483 3591 8314v20141116 NAD-dependent epimerase/dehydratase 1484 1484 3592 8315v20141116 NAD-dependent epimerase/dehydratase 1485 1485 3593 8318v20141116 hypothetical protein 1488 1488 3594 8331v20141116 hypothetical protein 1498 1498 3595 8335v20141116 hypothetical protein 3596 1502 3596 8473v20141116 ABC transporter-like protein 1521 1521 3616 8485v20141116 hypothetical protein 1532 1532 3618 8524v20141116 oxidoreductase 1570 1570 3619 8573v20141116 alkanal monooxygenase 1614 1614 3620 8579v20141116 hypothetical protein 1620 1620 3621 8922v20141116 response regulator receiver protein 3641 1749 3641 9277v20141116 transposase 1821 1821 3684 9290v20141116 diguanylate cyclase 1834 1834 3685 9309v20141116 XRE family transcriptional regulator 3687 1847 3687 9777v20141116 hypothetical protein 1934 1934 3729 10194v20141116 RTX toxins and related Ca2+-binding 1954 1954 3783 protein 10335v20141116 hypothetical protein Mnod_7733 2033 2033 3794 10354v20141116 Hypothetical protein 3795 2048 3795 10358v20141116 hypothetical protein 2050 2050 3797 12071v20141116 None 2288 2288 4101 12161v20141116 hypothetical protein 4103 2360 4103 MexAM1_META1p3214 14172v20141116 Fis family transcriptional regulator 2469 2469 4343

REFERENCES FOR EXAMPLE 6

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Ecol. 62: 243-250. -   17 -   6. Green, P. N. 2005. Methylobacterium. In Brenner, D. J., N. R.     Krieg, and J. T. Staley (eds.). “Bergey's Manual of Systematic     Bacteriology. Volume two, The Proteobacteria. Part C, The alpha-,     beta-, delta-, and epsilonproteobacteria.” Second edition. Springer,     New York. Pages 567-571. -   7. Green, P. N. 2006. Methylobacterium. In Dworkin, M., S.     Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.).     “The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5.     Proteobacteria: Alpha and Beta Subclasses.” Third edition. Springer,     New York. Pages 257-265. -   8. Holland, M. A. 1997. Methylobacterium and plants. Recent. Res.     Devel. in Plant Physiol. 1: 207-213. -   9. Holland, M. A., and J. C. Polacco. 1994. PPFMs and other covert     contaminants: Is there more to plant physiology than just plant?     Annu Rev. Plant Physiol. Plant Mol. Biol. 45: 197-209. -   10. Kutschera, U. 2007. Plant-associated methylobacteria as     co-evolved phytosymbionts. A hypothesis. Plant Signal Behav. 2:     74-78. -   11. Lidstrom, M. E. 2006. Aerobic methylotrophic prokaryotes. In     Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E.     Stackebrandt (eds.). “The Prokaryotes. A Handbook on the Biology of     Bacteria. Volume 2. Ecophysiology and biochemistry.” Third edition.     Springer, New York. Pages 618-634. -   12. Madhaiyan, M., S. Poonguzhali, H. S. Lee, K. Hari, S. P.     Sundaram, and T. M. Sa. 2005. Pink-pigmented facultative     methylotrophic bacteria accelerate germination, growth and yield of     sugarcane clone Co86032 (Saccharum officinarum L.) Biol. Fertil.     Soils 41: 350-358. -   13. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H.     Chung, J. Yang, S. Sundaram, and T. Sa. 2004. Growth promotion and     induction of systemic resistance in rice cultivar C0-47 (Oryza     sativa L.) by Methylobacterium spp. Bot. Bull. Acad. Sin. 45:     315-324. -   14. Madhaiyan, M., S. Poonguzhali, and T. Sa. 2007. Influence of     plant species and environmental conditions on epiphytic and     endophytic pink-pigmented facultative methylotrophic bacterial     populations associated with field-grown rice cultivars. J Microbiol     Biotechnol. 2007 October; 17(10): 1645-54. -   15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The     aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43:     159-271. -   16. Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., De     Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C., and     Dreyfus, B. 2001. Methylotrophic Methylobacterium Bacteria Nodulate     and Fix Nitrogen in Symbiosis with Legumes. Jour. Bacteriol.     183(1):214-220, -   17. Sy, A., A. C. J. Timmers, C. Knief, and J. A. Vorholt. 2005.     Methylotrophic metabolism is advantageous for Methylobacterium     extorquens during colonization of Medicago truncatula under     competitive conditions. Appl. Environ. Microbiol. 71: 7245-7252. -   18. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of     Escherichia coli: Partial purification and some properties. J. Biol.     Chem. 218: 97-106. -   19. Vogel, H. J. 1956. A convenient growth medium for Neurospora     (Medium N). Microbial Genet Bull 13: 42-43 -   20. Whittenbury, R., S. L. Davies, and J. F. Wilkinson. 1970.     Enrichment, isolation and some properties of methane-utilizing     bacteria. J. Gen. Microbiol. 61: 205-218. -   21. Vuilleumier S, Chistoserdova L, Lee M C, Bringel F, Lajus A,     Zhou Y, Gourion B, Barbe V, Chang J, Cruveiller S, Dossat C, Gillett     W, Gruffaz C, Haugen E, Hourcade E, Levy R, Mangenot S, Muller E,     Nadalig T, Pagni M, Penny C, Peyraud R, Robinson D G, Roche D, Rouy     Z, Saenampechek C, Salvignol G, Vallenet D, Wu Z, Marx C J, Vorholt     J A, Olson M V, Kaul R, Weissenbach J, Medigue C, Lidstrom M E.     Methylobacterium genome sequences: a reference blueprint to     investigate microbial metabolism of C1 compounds from natural and     industrial sources. PLoS One. 2009; 4(5):e5584. doi:     10.1371/journal.pone.0005584. Epub 2009 May 18. PubMed PMID:     19440302; PubMed Central PMCID: PMC2680597. -   22. Marx C J, Bringel F, Chistoserdova L, Moulin L, Farhan U I Haque     M, Fleischman D E, Gruffaz C, Jourand P, Knief C, Lee M C, Muller E     E, Nadalig T, Peyraud R, Roselli S, Russ L, Goodwin L A, Ivanova N,     Kyrpides N, Lajus A, Land M L, Medigue C, Mikhailova N, Nolan M,     Woyke T, Stolyar S, Vorholt J A, Vuilleumier S. Complete genome     sequences of six strains of the genus Methylobacterium. J Bacteriol.     2012 September; 194(17):4746-8. doi: 10.1128/JB.01009-12. PubMed     PMID: 22887658; PubMed Central PMCID: PMC3415506. -   23. Knief C, Frances L, Vorholt J A. Competitiveness of diverse     Methylobacterium strains in the phyllosphere of Arabidopsis thaliana     and identification of representative models, including M. extorquens     PA1. Microb Ecol. 2010 August; 60(2):440-52. doi:     10.1007/s00248-010-9725-3. Epub 2010 Aug. 11. PubMed PMID: 20700590.

The inclusion of various references herein is not to be construed as any admission by the Applicants that the references constitute prior art. Applicants expressly reserve their right to challenge any allegations of unpatentability of inventions disclosed herein over the references included herein.

Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.

Although the materials and methods of this invention have been described in terms of various embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 

What is claimed is:
 1. A method for improving tomato production, said method comprising applying a composition comprising Methylobacterium to a tomato plant, a part thereof, or to a tomato seed, wherein said composition comprises: (i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion having Methylobacterium grown therein; (iii) a Methylobacterium that has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594; or (iv) a Methylobacterium selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof; and wherein said tomato plant or tomato plant grown from said seed exhibits a trait improvement selected from the group consisting of an increased rate of root growth, leaf growth, seedling growth, seed production, fruit production, scion production, rootstock production, and/or increased total biomass when compared to an untreated control tomato plant or a control tomato plant grown from an untreated seed, thereby obtaining improved tomato production.
 2. The method of claim 1, wherein said composition comprises Methylobacterium at a titer of about 1×10⁶ CFU/gm to about 1×10¹⁴ CFU/gm for a solid composition or at a titer of about 1×10⁶ CFU/mL to about 1×10¹¹ CFU/mL for a liquid composition containing the solid substance or for the emulsion.
 3. The method of claim 1, wherein said Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594.
 4. The method of claim 3, wherein the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table
 4. 5. The method of claim 1, wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof.
 6. The method of claim 1, wherein said applied composition coats or partially coats said tomato plant or a part thereof, or said seed.
 7. The method of claim 1, wherein the method further comprises: (i) growing said tomato plant or tomato plant grown from said seed; and/or (ii) harvesting seedlings, rootstock, scions, fruit, or seed from said tomato plant or tomato plant grown from said seed.
 8. The method of claim 1, wherein the solid substance with adherent Methylobacterium is not a substance that promotes growth of resident microorganisms on the tomato plant, the part thereof, or the tomato seed.
 9. The method of claim 1, wherein the composition comprises an agriculturally acceptable adjuvant and/or excipient.
 10. The method of claim 1, wherein said composition is applied to said tomato plant, part thereof, or seed in a hydroponic solution.
 11. The method of claim 1, wherein said composition is depleted of substances that promote growth of resident microorganisms on said plant or seed. 12.-23. (canceled)
 24. A composition comprising: (i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion with Methylobacterium grown therein or contained therein; or (iii) a Methylobacterium; wherein said Methylobacterium has at least one gene encoding at least one protein that is orthologous to a protein having an amino acid sequence of SEQ ID NO: 1-4594 or wherein the Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 4, and wherein said composition further comprises an agriculturally acceptable adjuvant and/or excipient or wherein the composition comprises a hydroponic solution of man-made origin.
 25. The composition of claim 24, wherein the composition is depleted of substances that promotes growth of resident microorganisms on a plant or seed.
 26. The composition of claim 24, wherein said substance that promotes growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorous source, a sulfur source, a magnesium source, and combinations thereof.
 27. The composition of claim 24, wherein the solid substance with adherent Methylobacterium grown thereon has a Methylobacterium titer of at least about 5×10⁸ CFU/gm to at least about 1×10¹⁴ CFU/gm.
 28. The composition of claim 24, wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0037 (NRRL B-50941), NLS0066 (NRRL B-50940), and derivatives thereof.
 29. (canceled)
 30. A tomato plant part or tomato seed that is coated or partially coated with the composition of claim
 24. 31. A tomato plant part or tomato seed that is immersed or partially immersed in the composition of claim
 24. 32. (canceled) 